ANIMAL CLONING AND GENETIC MODIFICATION: AP ROSPECTIVE STUDY by Inkibj4H

VIEWS: 15 PAGES: 75

									         ANIMAL CLONING AND GENETIC MODIFICATION: A
                    PROSPECTIVE STUDY




                                               Report 1 to
              Institute for Prospective Technological Studies (IPTS)
                                                     Seville

                                                July 2005

                          R&D and commercialisation activities




Bruce Whitelaw1
Cecilia Oram2
Chris Warkup2
Ann Bruce3

1
    Roslin Institute, Roslin, Midlothian, Scotland
2
    Genesis Faraday Partnership, Roslin BioCentre, Roslin, Midlothian, Scotland
3
 Innogen (ESRC Centre for Social and Economic Research on Innovation in Genomics), University of
Edinburgh, High School Yards, Edinburgh, Scotland
                                 Acknowledgements
We would like to thank Moyra Forrest from Innogen for her help with the bibliographic review,
Catherine Lyall from Innogen for her help with searching literature and Eileen Mothersole for
contributing her secretarial skills. In addition we would like to thank all respondents of the
survey and interviewees contacted in the context of the project. Prof John Woolliams of
Roslin Institute and Dr Alan Tinch are thanked for their assistance with the economic
modelling related to cloning in pigs.
                                                   List of contents
Executive Summary .............................................................................................................. 1
SECTION 1 INTRODUCTION ............................................................................................... 4
   1.1 Introduction.................................................................................................................. 4
SECTION 2 OVERVIEW OF RESEARCH ACTIVITIES WORLDWIDE ................................. 5
   2.1 Introduction.................................................................................................................. 5
   2.2 Technical background.................................................................................................. 5
      2.2.1 Methods of producing cloned animals ................................................................... 5
      2.2.2 Methods of producing GM animals........................................................................ 8
      2.2.3 Methods of producing GM and cloned animals ....................................................12
      2.2.4 Summary: State-of-the-art....................................................................................14
      2.2.5 Bibliometric analysis ............................................................................................15
      2.2.6 Cloning ................................................................................................................15
      2.2.7 GM.......................................................................................................................17
      2.2.8 Cloning and GM ...................................................................................................18
   2.3 Future vision ...............................................................................................................19
      2.3.1 ES cells................................................................................................................19
      2.3.2 What species and to what applications? ..............................................................19
   2.4 Public versus private research ....................................................................................21
   2.5 Main drivers for research ............................................................................................21
SECTION 3: OVERVIEW OF COMMERCIALISATION ACTIVITIES WORLDWIDE .............22
   3.1 Introduction.................................................................................................................22
   3.2 Methodology ...............................................................................................................22
   3.3 Inventory of the companies active in the GM and/or cloned animal sector ..................23
   3.4 Summary of products in the pipeline ...........................................................................26
      3.4.1 Food production ...................................................................................................26
      3.4.2 Molecular pharming .............................................................................................34
      3.4.3 Xenotransplantation .............................................................................................39
      3.4.4 Pet sector ............................................................................................................40
      3.4.5 Sporting animals ..................................................................................................41
      3.4.6 Endangered species ............................................................................................41
      3.4.7 Other possible applications ..................................................................................42
   3.5 Comparison of economic structure of industry sectors ................................................42
      3.5.1 Food production ...................................................................................................42
      3.5.2 Molecular pharming .............................................................................................43
      3.5.3 Xenotransplantation .............................................................................................43
      3.5.4 Pet sector ............................................................................................................43
      3.5.5 Sporting animals ..................................................................................................43
      3.5.6 Endangered species ............................................................................................43
SECTION 4 TECHNO-ECONOMIC BARRIERS TO GM AND CLONING .............................44
   4.1 Main technical barriers................................................................................................44
   4.2 Main barriers to commercialisation .............................................................................45
      4.2.1 Cloning ................................................................................................................45
      4.2.2 GM.......................................................................................................................46
SECTION 5 COMPARISON OF EU WITH NON-EU COMPETITORS ..................................48
   5.1 Current research activities in the EU...........................................................................48
      5.1.1 Summary of EU position ......................................................................................48
      5.1.2 Summary of international perspective ..................................................................48
   5.2 Current commercialisation activities in the EU ............................................................48
   5.3 Non-EU countries closest to commercialising GM and cloned animals .......................49
SECTION 6 REFERENCES .................................................................................................50
   6.1 References .................................................................................................................50
   6.2 Bibliography................................................................................................................52
SECTION 7 GLOSSARY ......................................................................................................54
SECTION 8 APPENDICES ..................................................................................................58
   8.1 Appendix 1 .................................................................................................................58
   8.2 Appendix 2 .................................................................................................................60
   8.3 Appendix 3 .................................................................................................................64
   8.4 Appendix 4 .................................................................................................................70
                                                      List of figures
Figure 1: Publications relating to cloning in mammals (excluding mice), birds and fish .........16
Figure 2: Geographical distribution of publications relating to cloning in mammals (excluding
    mice), birds and fish ......................................................................................................17
Figure 3: Publications relating to GM in mammals (excluding mice), birds and fish ..............17
Figure 4: Geographical distribution of publications relating to GM in mammals (excluding
    mice), birds and fish ......................................................................................................18
Figure 5: GM and cloned animals for food production in the pipeline for three different time
    periods ..........................................................................................................................33
Figure 6: GM and cloned animals for pharmaceutical production in the pipeline for three
    different time periods .....................................................................................................34


                                                       List of tables
Table 1: Chronological History of cloning in different species ................................................ 6
Table 2: Dates of technological achievements in cloning and GM ........................................13
Table 3: First reports of GM livestock produced using cloning ..............................................19
Table 4: Inventory of companies active in the GM and/or cloned animal sector ....................23
Table 5: Description of GM and/or cloned animals for food production .................................33
Table 6: Annual yields of recombinant protein from various sources ....................................36
Table 7: Description of GM and/or cloned animals for pharmaceutical production ................36
Table 8: Description of GM and/or cloned animals for xenotransplantation...........................39
Table 9: Description of GM and/or cloned animals for pets ...................................................40
Table 10: Description of GM and/or cloned animals for sporting purposes ...........................41
Table 11: Key research priorities in cloning and GM .............................................................45
Executive Summary
The aim of this report is to summarise research and commercialisation activities in cloned
and GM animals worldwide. This report considers the technical and economic challenges
facing these developments as well as the relative competitive position of the EU, in so far as
this can be determined.
Research
The first mammalian species cloned using nuclear transfer was sheep, in 1996. Since then
several species have now been cloned including cow, goat, pig, horse, cat and most recently
dog. The most research, as measured by publications, has been on cattle. The second most
studied species is the pig, mostly for xenotransplantation applications, where cloning is
combined with genetic modification. The first genetically modified (GM) mammal was
produced in 1985 and GM pigs, sheep, cattle, goats, rabbits, chickens and fish have all been
reported. The main research use of GM techniques, as measured by publications has also
been for xenotransplantation.
A GM animal is one that has had ‘new’ DNA added to its germline. The introduction of new
genetic material can range from the addition of a given sequence to the replacement of a
given target sequence. Delivery of the new gene (or ‘transgene’) can be achieved by direct
injection or viral infection. With each approach the target can be the oocyte, sperm or
fertilised egg.
Cloned animals can be produced by nuclear transfer and efficiency of cloning has been
improved by several variations on this technique. Animals that are both cloned and GM can
be produced by the insertion of genetic material into a cell either by direct injection or using
viruses, followed by nuclear transfer from that cell to produce a clone. The first report of a
genetically modified and cloned animal (sheep) was in 1997. Much of the research in cloned
and GM animals has been accomplished by commercial establishments.
GM and cloning research can significantly advance our understanding of biology as well as
inform animal applications more directly. Research with animal embryonic stem cells and
cloning can be expected for example to inform surgical use of stem cells in human medicine.
Thus research on cloning and GM in animals can be viewed as underpinning important
developments in human health.
Commercialisation
The main potential commercial applications of cloned and GM animals include: food
production, production of pharmaceuticals (‘pharming’), xenotransplantation, pets, sporting
animals and endangered species. Of the 35 companies worldwide identified as working with
GM or/and cloned animals at the time of a survey undertaken for this report; 40% were
working in ‘pharming’, 15% in food production, 12.5% in pets, 10% in xenotransplantation,
10% in endangered species and 7.5% in other activities.
Cloned or GM animals already on sale include cloned pet cats, GM ornamental fish, cloned
horses and at least one rodeo bull. Individuals from some endangered species have been
cloned e.g. gaur, mouflon, banteng and African wildcat and cloning technology has been
applied to restoring endangered breeds of cattle.
A few new products are estimated to be at or near market and are likely to be commercially
available within the next 5 years, on the basis of evaluation by the commercial companies
themselves. Two pharmaceutical products from the milk of GM animals have completed
(Phase III and Phase II) clinical trials respectively and may be on the market in the EU in the
next few years.
Cloned livestock (especially pigs and cattle) are widely expected to be used within the food
chain somewhere in the world before 2010. It is likely that, within this timescale, it would not
be economic for cloned animals to be used directly for food or milk production, but that



                                               1
clones would be used as parents of slaughter pigs, beef cattle and possibly also milk-
producing dairy cows.
Faster-growing GM salmon are awaiting regulatory approval, principally for direct sale to fish
farming markets in N. America, Asia and S. America.
In the longer-term (beyond 2010) potential developments include the following:
Food production: GM fish other than salmon. For terrestrial livestock, a number of potential
applications include improving animal product composition, improved disease resistance,
reduced environmental impact and sterility.
Molecular pharming: A range of proteins produced from GM or GM and cloned cattle, goats
and chickens. Recombinant human mono- and polyclonal antibodies may be produced by
GM livestock beyond 2015.
Xenotransplantation: Proponents suggest that GM pig organ xenotransplantation to humans
is at least 10 years away
Technical barriers
Cloning
The main challenge for cloning animals is to improve efficiency and overcome problems of
Large Offspring Syndrome. Progress has been made with respect to both these, with some
commercial sources questioning the need for further improvement based on their internal
data for livestock species.
Cloning dogs proved more difficult than cloning livestock, but has now been achieved.
Cloning endangered species is difficult due to their unknown reproductive physiology and
difficulties in providing a reliable oocyte maturation environment. The lack of availability of
suitable recipients also constitutes a barrier.
GM
Major technical challenges with respect to GM include improving the accuracy of
incorporating transgenes (to avoid impacts on other parts of the genome or multiple copies of
genes) and improving efficiency. For GM fish, the lack of suitable regulatory elements to
control transgene expression is a challenge.
A major technical barrier in the food sector application of GM technology is the time required
to incorporate GM animals into existing breeding schemes. One of the technological barriers
to commercialisation of GM fish is sterilization efficiency, currently below 100%, which is
important if the fish are to be farmed in sea cages.
Technical barriers in the pharming sector include the long gestation period in cattle and the
limited number of antibodies which can be produced. Production of pharmaceuticals in
chicken eggs has been limited as the techniques for genetically modifying chickens have
proved to be more difficult.
Xenotransplantation remains a significant technical challenge, not least because multiple
transgenes are needed in order to overcome immune rejection mechanisms.
GM and cloned
Specific technical challenges for GM and cloned animals include the effects of ageing on
donor cells before nuclear transfer can be carried out and eliminating the need to include a
selection marker gene during the genetic modification step.
Main barriers for commercialisation
Cloning
The main barriers to commercialising cloning of food animals are consumer/food retailer
acceptance and, particularly in the USA, the regulatory uncertainty which currently exists.


                                              2
The regulatory uncertainty in the USA may be resolved within the next year or so. The
economic barrier to entry into animal cloning is likely to be low. Although it is not yet clear
what the costs of obtaining regulatory approval will be, these costs are likely to be
substantially lower than for GM livestock.
GM
The main barriers are perceived to be regulatory approval and consumer/food retailer
acceptance.
Regulatory approval of pharmaceutical products from GM animals is seen as the principal
hurdle followed by the time taken to get the product to market. Some of these barriers are
common to all pharmaceutical products, but there may be additional uncertainties to be
resolved for products from GM animals.
A barrier to attracting venture capital to start-up businesses working in this area of science is
the controversial nature of the work and the complicated IP situation for cloning technology.
European competitiveness
Research
Cloning research is undertaken primarily in USA, Far-East and EU countries. Although a late
entrant into the cloning research field, the Far-East may be rapidly becoming the major
research player. The majority of GM research has been carried out in the USA and Europe,
with Europe initially displaying a clear lead over the rest of the world. The majority of GM fish
effort is in the Far East and USA. As with cloning, the Far-East is a late entrant into this area
of research but is fast catching up.. If Europe aims to be a major player in cloning or GM
research then it will have to focus on specific aspects of this subject and to co-ordinate its
currently fragmented research effort (as is being done in the USA for example through
initiatives such as the National Swine Resource Centre).
Commercialisation
We have identified 35 companies worldwide involved in producing GM animals, cloned
animals or both GM and cloned animals. Of these, 63% are in N. America, 14% in Europe
and 11% in Asia.
Within the EU, France is the country which seems to be most actively commercialising GM
and cloned animals. French companies are applying GM and cloning to pharming, sporting
and endangered animal sectors. The Netherlands and UK each have one company active in
the pharming sector.
In terms of number of companies and scale of activity the USA has the most. Canada is
second but with significantly less activity than the USA. We should however note that we
experienced difficulty in obtaining information from companies in the Far East.
Non-EU countries are applying GM and cloning to a broad spectrum of possible application
sectors. The most significant difference between EU and non-EU countries is the focus of
non-EU countries on the application of GM and cloning to food production. In contrast the
companies in EU countries are applying GM and cloning principally to pharming but also to
sporting or endangered animals.




                                               3
SECTION 1 INTRODUCTION
1.1 Introduction
Since the development of cloned animals will facilitate the production of GM animals and
many of the applications of cloning (such as xenotransplantation) will depend also on the use
of genetic modification, these two technologies are investigated together. The purpose of this
study was to investigate the current status of developments in cloned and GM animals and
more specifically to provide a comprehensive picture of R&D and commercial activities
involving animal cloning and/or GM and their products world-wide; and a projected pipeline of
products for the next 5-10 years. It should be stressed that, in relation to commercialisation
activities, this study is very much a snapshot in time. Many of the companies involved in GM
and cloning developments in animals are small companies and they operate in an area of
significant commercial risk.
Regulatory, trade and socio-economic aspects are further considered in reports 2 and 3.
Cloned and GM animals can be used for a wide range of applications including food
production, molecular pharming, xenotransplantation, the pet sector, sporting animals and
endangered species. Development of animals to model human diseases and to investigate
gene function in model animals have however been specifically excluded from this report.
Within this report the term ‘cloning’ should be understood to mean somatic cell nuclear
transfer (SCNT) unless otherwise stated and the term ‘animal’ should be understood to refer
to non-human animals.
This report is in five sections:
   Section 1 is an introduction
   Section 2 maps research and development activities worldwide involving cloned and GM
    animals
   Section 3 maps out commercialisation activities.
   Section 4 considers the technical and economic barriers to developments with respect to
    cloned and GM animals and finally;
   Section 5 considers the position of the EU in comparison to non-EU competitors.
This study was conducted March-July 2005. The scientific aspects were investigated by staff
at Roslin Institute, the commercialisation activities by staff at Genesis Faraday Partnership
and co-ordination was provided by the Innogen Centre. The methodology consisted of
literature and web surveys supplemented by interviews, bibliometric analysis based on
Articlefirst and Web of Science, and questionnaires, as outlined in each of the relevant
sections. Additionally, useful interchange of information took place with the Specific Support
Action “Farm animal cloning and the public” and in a 2-day workshop held in Seville in June
2005, co-organised by the IPTS and the above mentioned SSA.




                                              4
SECTION 2 OVERVIEW OF RESEARCH ACTIVITIES WORLDWIDE
2.1 Introduction
This section aims to convey an overview of on-going research activities and technological
developments in animal cloning and/or genetic modification worldwide, including national and
EU funded programs. Specific emphasis is placed on short and medium term visions (5 and
10 year span).
GM and Cloning are intertwined in both public perception and application, although they
represent different technical methods in animal biology. This will be described using
examples and species differences. Applications in large animals are mainly restricted to
commercial projects due to the cost and effort involved. This aspect will be developed
through discussion of the drivers and incentives for research in this area and contrasted to
the main technical barriers for research. In addition, the position the EU holds in the global
arena will be presented.
This report is based on original data and quantitative information on GM and/or cloned
animals, their offspring and derived products worldwide. It also builds on publicly available
information in the literature, relevant databases (as outlined in Appendix 1) and expert
interviews. The expert interviews were performed face to face when possible, with a few by
phone, and by email. In total 34 experts in the cloning and GM field were contacted. The
geographical distribution of the experts was 23 from the EC, eight from the USA and three
from Australasia (including one from China).
To emphasise, this section deals with the research aspects of cloning and GM. By definition
there is a strong focus on technical and proof-of-concept aspects of cloning and GM.
2.2 Technical background
2.2.1 Methods of producing cloned animals
Cloning in this context relates to the production of animals through transfer of the genetic
material from one donor cell to a recipient unfertilised oocyte that has had its nuclear DNA
removed (enucleation). This process is also known as somatic cell nuclear transfer (SCNT).
Through the use of several individual cells from a given unique source and an equivalent
number of recipient oocytes, several cloned animals can be produced.
i. Overview
Although in use and robust, the standard method for producing transgenic animals apart from
mice is inefficient, not exact and costly. These limitations drove researchers to seek other
methods. Half-a-century ago, the background to animal cloning was demonstrated by
transplanting nuclei from the blastula of the frog to an enucleated oocyte, obtaining a number
of normal embryos in the process. Subsequently, nuclei from various types of cell were
transplanted to an oocyte that has been subjected to ultraviolet radiation to destroy the
peripheral chromosomes. Again embryos were produced. These results establish the
important principle that while animal cells become committed to their fate as development
proceeds, the nuclei of most cells still retain all the genetic information required for the entire
developmental programme and can be reprogrammed by the cytoplasm of the oocyte to
recapitulate (restart) development (reviewed by Campbell et al. 1996).
This ability of differentiated cells to be reprogrammed was dramatically demonstrated in 1997
through the publication by Wilmut and colleagues of the method that generated ‘Dolly’ the
sheep (Wilmut et al. 1997). The birth of animals from differentiated foetal and adult cells also
reinforces previous speculation that by inducing donor cells to become quiescent (cell-cycle
resting state) it will be possible to obtain normal development from a wide variety of
differentiated cells. In all species, it appears that the earlier the developmental stage at which
nuclei are isolated, the greater their potential to be reprogrammed. Nuclear transplantation
can be used to generate clones of animals with the same genotype by transplanting many



                                                5
somatic nuclei from the same individual into a series of enucleated oocytes - cloning. In
addition, by using a genetically modified donor cell a transgenic animal can be produced.
Since the first use to produce sheep, a wide range of animals has been cloned including
pigs, cattle, goats, rabbits, mice, rats, mules and horses and fish. In addition, various breeds
within some of these species have been cloned.
Table 1: Chronological History of cloning in different species
 Date first cloned          Mammalian species
 1996                       Sheep
 1998                       Cow
 1998                       Mouse
 1999                       Goat
 2000                       Pig
 2002                       Rat
 2002                       Rabbit
 2003                       Mule
 2003                       Horse
 2004                       Cat
 2005                       Dog


ii. Nuclear transfer
Nuclear transfer (NT) is one of the most interesting and challenging areas of science to
emerge in recent years. This procedure involves the transfer of a nucleus from a donor cell to
an oocyte that has had its genetic material removed. The donor cell is somatic in origin and
could be an embryonic or adult cell (so-called somatic cells) rather than a germ cell (sperm
or egg) normally involved in reproduction. The donor nucleus is introduced by promoting
fusion between the oocyte and a somatic cell and brief electric pulse is often used to achieve
this as it also activates embryonic development by stimulating the mobilization of calcium
ions.
The success rate of nuclear transfer consistently falls within the range of 1 to 5% (of injected
fertilised eggs) showing that at the moment the technique has an inherent inefficiency,
although some commercial organisations now claim up to 30%. There are many theories as
to the reason for this inefficiency but, at present, these remain speculative. Embryos
transferred to surrogate mothers are lost in an unusual pattern, as the losses occur all the
way through pregnancy and continue after parturition. There have also been defects in many
cloned animals, with reports of defective immune systems, cardiovascular problems, obesity,
urogenital abnormalities and others, many of which are fatal. Indeed, ‘Dolly’ herself was
suffering from arthritis but no one knows whether this was due to the nuclear transfer
process or a chance event in her life; ‘Dolly’ died of natural causes, a Jaagsiekte virus
infection.
Obviously the genotype (excluding mitochondrial DNA) of the cloned animal is dependent on
that of the donor nuclei. If this donor cell carries a transgene (introduced genetic material in
the form of a DNA sequence) then the resulting animal will be transgenic. The production of
genetically modified and cloned animals was first demonstrated for random transgene
integration in 1997 (Schnieke et al. 1997) and using a method that targeted the introduced
gene to a specific site on the chromosome three years later (McCreath et al. 2000).



                                                6
Alternatively, genetic selection of the nuclear donor can lead to production of genetically
modified clones of the desired gender and appropriate production traits.
iii. Variations on nuclear transfer
Cloning to-date involves the use of a donor nucleus and recipient oocyte. As yet an unknown
component of the oocyte is able to re-programme the donor genetic material such that it
starts developing as an embryo and subsequently grows into an adult animal. If we were able
to understand the signal provided by the oocyte – identify the components that are able to
cause re-programming – we could perform the procedure without the use of an oocyte. In
addition, it is possible that the donor genome could be provided as a molecular compound
rather than a cell. As yet, this appears a long way off. Currently the source of all donor cells
has been from somatic cell populations within the respective animal.
In nuclear transfer reconstructed embryos, the co-ordination of donor nuclear and recipient
cytoplasmic cell cycle phase is essential to maintain ploidy and prevent DNA damage. A
critical factor in the success of nuclear transfer was the quiescent state of the donor cells in
culture and the recipient enucleated egg allowing synchronization between the donor and
recipient cell cycles. For the production of ‘Dolly’, this was achieved by lowering the level of
serum in the culture medium, causing the cells to withdraw from their normal cell cycle
activity due to lack of growth factors. However, the stage of the cell cycle at the time of
reconstruction and the method of reconstruction may also have a significant impact on the
subsequent development of the embryo and foetus through a number of other mechanisms
that remain to be characterised.
Variations with respect to the donor genetic material have been developed. Perhaps the
most promising has been the remodelling of the somatic nuclei in vitro prior to cloning by
nuclear transplantation – termed chromatin transfer. One approach involves permeabilisation
of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of
nuclear factors solubilised during chromosome condensation (Sullivan et al. 2004). This
treated cell is then used as the donor in the cloning step. There are claims that this approach
can overcome the Large Offspring Syndrome often seen in cloning experiments.
With regard to the actual procedure, numerous variations also exist including for example,
hand-made cloning (Vajta et al. 2005). This method differs from the more established
protocols by involving removal of the zona pellucida (a jelly-like substance surrounding the
early embryo up to blastocyst stage) prior to enucleation and fusion, resulting in a limited (or
no) requirement for micromanipulators. These variations may have individual technical
advantages, some of which lend themselves to mechanisation of the cloning procedure, but
they all still involve the use of a donor somatic cell and recipient oocyte.
iv. Example applications
The majority of research using cloning falls under two themes. The first is simply attempts to
improve the method. Secondly, cloning has been used to investigate early embryo
development and the reprogramming of the genome, including epigenetics. Much knowledge
has been gained through these studies.
In addition, research in cloning has been used to demonstrate the potential application of this
technology. There have been three targets: cloning of genetically superior animals destined
for animal breeding regimes; cloning of pet companion animals; and cloning of endangered
or rare species.
v. General issues
Animal cloning has been with us for a decade. In this time it has transformed how we view
cell biology, while significantly raising the biology debate within society. The importance of
somatic cell nuclear transfer goes far beyond the scope of replicating animal genotypes. It is
an invaluable experimental tool to address fundamental scientific issues such as genetic
plasticity, cell differentiation, genome structure and function (including epigenetics) and


                                               7
genome manipulation. For these reasons the importance of cloning is not for what it can
achieve but for the technical support it can provide to animal and biomedical research. The
latter includes the study of epigenetics, cancer and stem cell biology, cell therapy and
regenerative medicine. For example, embryos produced by nuclear transfer from a patient's
somatic cell offer one potential source of embryonic stem cells for treatment of human
degenerative diseases.
Inter-species cloning has become topical. This approach to mixing the species of origin of the
donor and recipient cell offers some advantages for exploring scientific questions and
provides tools to understand the bio-risk aspects of cloning. However, since the resulting
animal would be a chimera of nuclear and mitochondrial DNA, its general use is currently
being debated. It should also be considered that if embryonic stem (ES) cells were derived
from animals these could provide a donor source that allows more efficient procedures. This
would also have benefits for genetic modification. There is some general agreement within
the cloning and GM scientific community, that if one scientific priority is to be identified, then
the derivation of stem cells from species other than the mouse is arguably the most important
(major theme within 23 EU scientists interviewed).
2.2.2 Methods of producing GM animals
A GM animal is one that has had ‘new’ DNA added to its germline. Following the introduction
of the transgene it is passed from one generation to the next in a well understood manner.
The introduction of new genetic material can range from the addition of a given sequence to
the replacement of a given target sequence. As such, the key aspect is the delivery of the
‘new’ DNA or transgene. This can be achieved by direct injection or viral (vector) infection.
With each approach the target can be the oocyte, sperm or zygote (fertilised egg).
i. Overview
From a historical perspective the first GM mammals were transgenic mice (Gordon et al.
1988). It is still true today that the majority of transgenic mammals are mice and furthermore
that the majority of technical developments are initially established in this rodent species.
The pioneering work in the early 1980’s set the stage for the first transgenic livestock in 1985
(Hammer et al. 1985). All of this early work involved the delivery of the DNA transgene by
direct pronuclear injection of zygotes. It was 20 years before this technology was truly
furthered, with the demonstration that lentiviral vectors allow transgene delivery at previously
unachievable rates (Whitelaw, 2004). This later technology has now been applied to mice,
rats, pigs, sheep and cattle.
Attempts to transform the germline of birds have been developed, although the technical
hurdles have differed from those overcome for transgenic mammals.
In parallel, germline transformation of fish has been widely achieved.
ii. Pronuclear injection
Direct microinjection of DNA was the first strategy used to generate transgenic mice. The
technique involves the injection of DNA into one of the pronuclei (which contain the genetic
material of the host in the form of chromosomes) of the zygote. Either short- or long-
sequence transgenes can be delivered. The procedure requires specialized microinjection
equipment and considerable dexterity from the handler. The technique is reliable, although
the efficiency varies, so that 5-40% of mice developing from manipulated oocytes contain the
transgene.
In general this efficiency drops with increasing size of transgene fragment. In livestock, the
efficiency is lower for a number of reasons. First, the embryos are more fragile than those of
the mouse making manipulation less efficient. Secondly, for most livestock species,
visualisation of the pronuclei is difficult due to the presence of opaque lipid droplets in the
cytoplasm. This often necessitates a centrifugation step and care is required not to damage
the zygote. Pronuclear injection in domestic animals was first demonstrated in pigs.


                                                8
Subsequently, transgenic cattle, goats and sheep have been produced using this method
(Clark and Whitelaw, 2003).
Founder animals can be mosaic for the transgene (i.e. only some cells carry the transgene),
but once the transgene is transmitted through the germ line it tends to be stably inherited
over many generations. The transgene tends to form head-to-tail arrays prior to integration
although other arrangements are detected at integrated loci (it is not understood why these
arrangements occur), and the copy number varies from a few copies to hundreds. Although
not common, extensive deletions and rearrangements of the host DNA may often
accompany transgene integration.
iii. ICSI
Originally developed to overcome infertility in humans, the injection of sperm heads directly
into the cytoplasm of the oocyte termed intra-cytoplasmic sperm injection (ICSI) can be used
to generate transgenic animals. The DNA transgene can be chemically-bound to the sperm
head or simply co-injected with the sperm into the cytoplasm. This approach is appealing but,
as yet, has not been widely taken up. This lack of support is largely due to issues of
variability in the procedure. New developments include combining ICSI with recombinase
enzymes to facilitate transgene integration (Kaneko et al. 2005) and the delivery of large
transgene fragments, e.g. based on yeast artificial chromosomes. The latter approach can
provide reasonably high efficiencies of up to 35%, in contrast to pronuclear injection which at
best gives 5% in livestock.
iv. Viral vectors
Building on the existing use of replication-defective viral vectors for gene therapy in humans,
several vector designs have been applied to transgenesis in livestock. Vectors derived from
retroviruses – either the oncoretrovirus or lentivirus – have proved most effective. These
viruses are complex in genetic make up, and their life-cycle involves integration into a host
genome. The vectors based on retroviruses are engineered through the removal of
numerous key DNA sequences such that they can integrate but are then unable to become
mobile again. Thus they cannot replicate, a prerequisite for a successful viral vector. In
addition, some reports claim success with adenovirus vectors (Tsukui et al. 1996) but this
method has not seen widespread uptake.
Recombinant retrovirus vectors provide a natural mechanism for stably introducing DNA into
the genome of animal cells. Retroviruses are able to infect early embryos, so recombinant
retroviral vectors can be used for germline transformation. An advantage over the
microinjection technique is that only a single copy of the retroviral provirus is integrated at a
given locus, and the genomic DNA surrounding the transgenic locus generally remains intact.
Animals can, however, carry more than one integration event at different chromosomal
locations. The infection of preimplantation embryos with a recombinant retrovirus is
technically straightforward and, once the infected embryos are implanted in the uterus of a
foster mother, can lead to germ line transmission of the transgene.
The primary advantage of lentiviral vector is the spectacular efficiency they afford. Using the
standard microinjection procedure, between 1-5% of injected eggs will give rise to a
transgenic founder animal which translates into between 5-20% of births that are transgenic.
Through the use of lentiviral vectors, as long as a high enough virus titre is produced,
upwards of 30% of the injected eggs result in a transgenic founder animal, with up to 90% of
births being transgenic. This has two immediate effects: firstly, to reduce the number of
animals used, constituting a major welfare benefit; and secondly, a substantial reduction in
cost. The latter should result in an increase in publicly funded opportunities leading to an
increased use of the technology in the academic world. The use of viral vectors is not
expected to be taken up by the commercial sector, due to public suspicion of anything to do
with viruses. This restriction on the use of viral vectors may diminish if the use of virus-based
delivery systems becomes commonplace in medical therapies.



                                               9
The major disadvantage of viral vectors is that they are limited in the amount of DNA they
can carry; about 4.0 kb for an oncoretrovirus, 7.5kb for adenovirus. Thus they can only carry
small transgenes, limiting their application. In addition, use of oncoretroviruses has been
restricted by the commonly observed methylation-associated transgene silencing and their
propensity to affect expression of host genes that neighbour the site of transgene integration;
similar concerns are appearing in some studies using lentiviral vectors which can result in
weak expression patterns. If this occurs then host gene activity can be affected which could
be disadvantageous to the animal. There scientific community is currently attempting to
devise strategies to overcome these limitations.
v. Transgenic birds
The development of techniques for the genetic manipulation of poultry has lagged behind the
technology available in mammalian systems. Initially hope was pinned on the tried and tested
mammalian approach, that of microinjection but, given that access to the chicken egg is only
available after it has started to develop and thus comprises many thousands of cells, this has
met with little success. Nevertheless live transgenic birds can be produced by this route
demonstrating that manipulation of the chick zygote is possible but inefficient (Love et al.
1994).
Subsequently considerable progress has been made in deriving embryonic cells, and
demonstrating their ability to contribute to somatic chimeric birds is well-established.
However, germline transmission of gametes derived from genetically modified embryo cells
has not been described. Although transfer of primordial germ cells from a donor embryo to a
recipient and production of functional gametes from the donor-derived cells is possible,
genetic modification of primordial germ cells before transfer and their recovery through the
germline has not been achieved.
Very recently the use of lentiviral vectors for the production of transgenic chickens has been
evaluated and the efficiency of production of founder transgenic birds and of transduction of
the germ line was at least 10-fold higher than has been described using retroviral vectors
(McGrew et al. 2004). Furthermore, high-level transgene expression was observed and
maintained through subsequent generations.
Cloning has not been achieved for birds and it is not obvious how this technology would be
developed in birds.
vi. Transgenic fish
Gene transfer technology in fish has also lagged behind that of mammals but for different
reasons. Transgene delivery has not proved to be a limitation, rather the lack of suitable
transcription regulatory elements to control transgene expression. The first transgenic fish
carried transgenes driven by mammalian or viral regulatory elements, and their performance
varied considerably. This has now been overcome through the use of gene regulatory
sequences derived from fish species.
Like amphibians, the microinjection of DNA into fish oocytes and early embryos leads to
extensive replication and expression from unintegrated transgenes. Some of the DNA
integrates into the genome, leading to germ line transmission, and the production of
transgenic fish lines. There is now considerable effort to improve on this random integration
through the use of viral vectors or vectors based on transposable sequences such as
Sleeping Beauty transposon.
There has also been considerable effort to derive ES cells for fish species. However, this
remains a largely unattained goal. Although zebrafish embryo cell cultures that exhibit
characteristics of embryonic stem cells have been described, the successful contribution of
the cells to the germ-cell lineage of a host embryo has not been reported. Analogous cells
have been reported for medaka and some other species but again none have reliably




                                              10
achieved germ-line transformation. A range of fish have been genetically modified (see
section 3.3.1;i).
The development of GM fish has raised substantial concerns with regard to environmental
risk. Risks are further considered in Report 3, section 3.1.
vii. Example applications
The most established application of GM to animals is that of the bioreactor – animals that
produce proteins destined for biomedical use, usually from a transgene designed to target
protein production to milk. There have also been attempts to produce animals with altered
production characteristics, ranging from growth rate through altered product composition to
wool production, and as yet unsuccessful attempts to generate GM animals with enhanced
ability to combat disease. In birds the main application target is bioproduction of proteins, the
bird (all effort to date-has been with the chicken) becoming a bioreactor. Recent progress
with lentiviral vectors has produced the first real technical successes in this field. In fish the
main application has been to enhance growth rate, with some spectacular successes
achieved. However, history may deem the most significant to be the failed attempt to
generate fish as pollution monitors. These fluorescent fish have become the first
commercially available GM animals, attracting interest as glowing pets. More recently effort
has been applied to devise refined gene manipulation strategies across all species, using
viral or transposable vectors and recombinase systems.
viii. General aspects
Direct transgene delivery is associated with specific characteristics regardless of the precise
delivery route or vector. First, the transgene effectively integrates at random within the
genome. As a consequence, rare integration events can disrupt an endogenous gene.
Evidence from mouse studies indicates that this is a rare event, at least as evaluated in
hemizygous transgenic lines. Secondly, the transgene can integrate as a single-copy or
multiple-copy. There is no control over either of these aspects of the process.
It is possible to retrospectively reduce a multicopy transgene locus to a single copy locus.
This requires that the transgene is engineered in vitro to carry a single bacterial recombinase
site, e.g. LoxP. Then in the presence of the bacterial recombinase, e.g. Cre, which can be
transiently delivered to the oocyte or zygote, the locus recombines by deleting the internal
transgene copies leaving a single copy. The random arrangement of transgene units within a
multicopy locus, i.e. head-to-head or tail-to head or tail-to-tail, means that the recombinase-
based strategy will not always be successful. To-date, although successfully employed for
transgenesis in mice (Ristevski, 2005), the recombinase approach has not been applied to
larger mammalian species.
There are two general disadvantages of direct transgene delivery as a direct result of the
above characteristics. These apply to all species tested to-date. The first reflects the site of
integration. The activity profile for a given transgene can be modulated by where it has
integrated into the host genome. For example, if a transgene integrates next to a string
tissue-specific gene transcriptional enhancer, the desired transgene cell-type activity profile
may be affected by specificity of the enhancer rather than what was intended. Alternatively, if
a transgene integrates into heterochromatin, a region of the genome that is devoid of gene
activity, the transgene can be silenced (inactivated).
The second disadvantage is that most transgenes do not express on a copy-number basis,
i.e. more transgene copies do not necessarily mean more transgene activity. There are
exceptions to this, where a transgene incorporates a dominant transcription regulator,
sometimes called a locus control region. In specific cases the presence of such dominant
transcription sequences can result in copy-dependent transgene activity.
Combined, effects of site of integration and copy-number are often termed the position-effect.
The position-effect can range for complete transgene inactivity, through partial activity or



                                               11
inappropriate temporal or spatial activity, to the desired and expected expression profile. In
practice, the position-effect means that several individual transgenic lines have to be
generated and screened for the desired expression profile i.e. each transgenic line is based
on a unique animal.
The use of some viral vectors, e.g. those based on lentivirus, appear to overcome one of
these limitations in that they are associated with efficient and reliable transgene activity
profiles (although, more studies are required to confirm this positive attribute). It is not
understood why this is so, but it is anticipated that investigation of the chromatin structure
associated with integrated viral vectors will likely address this question.
2.2.3 Methods of producing GM and cloned animals
The technologies of cloning and GM can be combined. This occurs when the donor cell for
cloning is GM, i.e. transgenic. Thus the cloned animal produced will carry the transgene and
transmit the transgene to its offspring.
i. Rationale
The limitations of direct transgene delivery – integration site and copy-number combining
under the term position-effect – have been circumvented in mice through the use of
homologous recombination (DNA sequence specific recombination between two DNA
fragments) in ES cells and in livestock through the use of nuclear transfer (cloning). The
search for ES cells from species other than the mouse (and similar cells for humans) has not
been successful to date. This was one of the main drivers for alternative strategies which
lead to development of the nuclear transfer method. Being able to generate animals from
cells that had been manipulated in vitro was a significant breakthrough in animal
biotechnology.
ii. Embryonic stem cells
Embryonic stem (ES) cells are isolated from undifferentiated cells of the early embryo,
specifically from the inner cell mass of the blastocyst. This very peculiar cell type displays
three useful properties. First, an ES cell is capable of self-renewal. Given the appropriate
culture conditions ES cells will divide to produce more undifferentiated ES cells indefinitely
(or until the conditions change or become exhausted). Secondly, ES cells display a high
frequency of homologous recombination. This allows specific genetic modifications to be
engineered, and subsequently selected in such a way that only cells with this genetic change
are present within the test cell population. In this way a gene can be destroyed, replaced by
an alternative form of the gene or a completely different gene, single point mutations
incorporated or controlled chromosome rearrangements induced. Finally, ES cells have the
capacity to differentiate into the full range of embryonic tissues, and thus can be used to
generate an entire animal after incorporation into an early embryo.
Specific genetic changes can be induced into an ES cell, selected for in vitro, returned to the
early embryo usually at the blastocyst stage where the cells resume their normal program of
development resulting in the birth of a GM animal. The founder animal is usually a chimera
derived from the ES cell and the host blastocyst. The extent of chimerism varies from
individual to individual, and when the ES cell has contributed to the germline the resulting
second generation animals will be entirely GM.
Although there has been considerable interest in deriving ES cells from mammalian species
other than the mouse, and many claims made over the last two decades, none of these
reports have meet the definitive test of germline transmission. In addition, at the moment
evidence is emerging that pluripotent stem cells can be isolated from the testes. If robust,
this source of stem cells may offer new approached to the generation of GM animals.




                                              12
iii. History of cloned GM animals
The first transgenic animal to be produced by cloning with transgenic donor cells was ‘Polly’
in 1997 (Schnieke et al. 1997). This sheep carried a transgene designed to target expression
of human factor IX to milk. The transgene was randomly integrated into the host genome in
vitro. Three years later cloning was employed in gene targeting strategies (McCreath et al.
2000). In this way the procedure is analogous to the use of homologous recombination in
mouse ES cells. This is arguably one of the most common uses of cloning technology and to-
date transgenic sheep, pigs and cattle have been generated in this way.
Ironically even though the driving force behind the development of nuclear transfer in animals
was to provide a cell-base route to transgenesis, there have been only a handful of studies
exploiting this potential. There have been two interconnected reasons.
The first is that the process is technically demanding and inefficient. Considerable financial
support is required to undertake this methodology. Specifically, gene targeting in somatic
cells rather than embryonic stem cells is a challenge. The recombination frequency in
somatic cells is less than that of ES cells. In addition, somatic cells have a limited lifespan so
all the manipulative steps involved in a gene targeting event must be accomplished prior to
cellular senescence. Furthermore, dogma had it, justified on the basis of our understanding
of chromatin, that targeting of inactive (transcriptionally silent) genes or double targeting to
produce homozygotes was difficult.
There have been a few reports of double targeting events, but it is only recently that this
objective has been overcome. The reporting in 2004 of sequential gene targeting in cattle
(Kuroiwa et al. 2004) has provided a major step forward for this technology. The method
consists of sequential application of gene targeting by homologous recombination in
fibroblasts (somatic cells) and rejuvenation of cell lines by production of cloned foetuses.
Although technically a tour de force this is still hugely demanding technically and very
expensive. Several groups, including some in Europe, are currently attempting to repeat this
procedure.
Table 2: Dates of technological achievements in cloning and GM
 Event (first report)       Date              Research group                 Country
 GM cloned livestock        1997              PPL Therapeutics Ltd           UK
 Gene knock-in              2000              PPL Therapeutics Ltd           UK
 Gene knock-out             2001              Roslin Institute               UK
 Large transgene            2002              Kirin Brewery Co               Japan
 Sequential targeting       2004              Hematech LLC                   USA


One aspect of the above major events in cloned and GM animals is that most were
accomplished by commercial establishments.
Secondly, most studies to-date, have suffered from the Large Offspring Syndrome (LOS) and
developmental defects that result in foetal death. It is assumed that these defects are a
continuum of phenotypic responses to some, probably nutritional, effect of in vitro culture
rather than cloning per se, as LOS is seen for in vitro cultured embryos that have not been
cloned (Lonergan et al. 2003, Wrenzycki and Niemann 2003).
LOS relates to the often-observed birth of large offspring in cloning studies. Overgrowth
phenotypes across the species exhibit many common features, including alterations in organ
and tissue development, and placental anomalies. Imprinted genes have been implicated in
this syndrome and current understanding points to sub-optimal nutritional components of the
in vitro culture medium required during cloning (and other embryo manipulative processes).



                                               13
In addition, LOS is only a problem in the first generation cloned animal. There is no evidence
that the phenotype is transmitted to the offspring.
As culture media become more defined and the procedure more routine, at least in the larger
commercial breeding companies, the frequency of LOS is reducing. Alternatively,
modification of the donor genome and the chromatin encompassing it (chromatin
remodelling) has also been proposed to reduce the welfare issues. Compared with nuclear
transfer, chromatin transfer shows a trend towards greater survival of cloned animals (but
has only been performed in cattle so far; Sullivan et al. 2004).
Notwithstanding all this progress, it is not clear yet whether the limitation of LOS has been
truly overcome.
iv. Example applications
To-date the generation of GM animals using cloning has been applied to two goals. The first
one that has attracted a considerable amount of research is the generation of animal donors
for xenotransplantation. The second is for production of animal bioreactors. This latter
application is the closest to commercial reality with several products derived from transgenic
livestock in late clinical trials. Cloning by nuclear transfer has also been applied to fish,
specifically zebrafish, but it is not clear what advantage this offers over the already efficient
standard breeding strategies that can be employed in fish (particularly in view of the high
fecundity of fish).
v. General aspects
There are two further issues that should be considered in regard to the generation of cloned
GM (transgenic) animals, transgene design and donor cell senescence.
Most issues of transgene design are easily accommodated with current molecular biological
techniques. However, most transgene designs include a selection marker gene which allows
only the desired genetically modified cells within a large population of cells (most of which do
not carry the transgene) to be selected for. The non-transgenic cells die since they do not
harbour the selection marker gene, e.g. resistance to puromycin which causes inhibition of
the translation of mRNA into protein thus killing a cell. The presence of some selection
marker gene sequences has been associated with gene silencing events while there is also
some concern of generating animals expressing antibiotics. There are strategies available,
e.g. through the use of a recombinase system, that enable these sequences to be removed.
There is no reason why they cannot be applied to animals; they are applied routinely in mice.
The second issue of donor cell senescence has proved problematic. Somatic cells, the donor
cell type in cloning, have a limited lifespan in culture. This makes the process of gene
targeting, which involves an extended selection step during which non-targeted cells are
killed, difficult, i.e. by the time targeted cells have been selected and isolated, the cells have
senesced making them inappropriate donors for nuclear transfer. Attempts to overcome this
have included the use of telomerase to extend the life span of the target cell. Although some
success has been achieved this is not an ideal approach given that the resulting animal is
derived from altered cells. Recently, the issue has been largely overcome through the use of
sequential cloning and rejuvenation of cell lines through the production of a foetus (Kuroiwa
et al. 2004). This procedure remains to be repeated to test how robust it is. Nevertheless, it
appears that a major step forward has been achieved.
2.2.4 Summary: State-of-the-art
Scientists have been able to generate transgenic livestock for two decades. First through the
tried and tested but inefficient method of pronuclear injection, but only recently within the last
few years have major advances been achieved through the use of specific viral vectors. Not
only do lentiviral vectors allow efficient genetic modification but they also appear to allow
efficient transgene expression by apparently overcoming gene-silencing events. They do
have their limitations and further development is required.


                                               14
The desire for a cell-based system led to the development of nuclear transfer (cloning) in
1996 with the generation of the sheep ‘Megan’ and ‘Morag’ (Campbell et al. 1996). Morag is
still alive and is now nearly ten years old. This paved the way for gene-targeting, which was
achieved in 2000 (McCreath et al. 2000). Nevertheless, the generation of GM animals
through the use of cloning is still technically demanding. Last year, the development of a
sequential cloning protocol has again taken the field forward (Kuroiwa et al. 2000), although
this use of cloning is still very expensive.
Cloning has developed from this perspective into a tool in its own right. Since the first
demonstration in 1996 using embryonic cultured cells, then the following year with the
generation of ‘Dolly’ from adult mammary cells, cloning has been applied to several species.
Variations on the procedure and modification to the donor cell have been developed, while
recent claims suggest that the welfare issue of developmental defects might be overcome
through controlled in vitro culture conditions. Although research still progresses in the
academic arena, the commercial world has already taken this method onboard with the
future application in the hands of the regulators and commercial viability. Further information
on the technical aspects of cloning and GM animals can be found in the references listed
under the bibliography section of this report.
2.2.5 Bibliometric analysis
We have performed a web-based search of the scientific literature. This provides a flavour of
the reported achievements in cloning and GM but is not definitive. No one database is
exhaustive in its coverage of these topics, nor is there consistency in the use of terms.
Specifically we do not believe that there is full representation of the scientific literature for
countries in the Far-East, in particular China. Nevertheless, the data is sufficient in our
estimation to provide an overview of the progress in cloning and GM.
We searched the following web-database resources; Articlefirst on Firstsearch and Web of
Science. The former is limited in that neither URLs nor authors’ addresses are provided. The
latter did not give thorough coverage of key journals; these we searched through their
respective journal web pages (Nature, Nature Biotechnology, Nature Genetics, Nature
Medicine and EMBO Journal).
The search was restricted to English language publications, ignored GM/cloned mice and
insects, and focussed on mammals, birds and fish. Our remit was to focus only on the
scientific literature not grey literature. Where possible we identified from the authors
affiliations which country led the work, subdividing the field into cloning, GM or cloning and
GM. This was not always possible. A more detailed methodology is given in Appendix 1.
2.2.6 Cloning
We identified just over 500 publications. The vast majority, estimated at 90%, have public
funding support. This figure must be treated with caution, as the involvement of commercial
funding is not always transparent in the author affiliations. The majority of publications
address technical aspects of cloning and attempt to reduce the inefficiency and LOS. It is fair
to say that the key papers have come from both public and commercial funded research. We
have summarised the data below, and highlighted specific aspects of this study.




                                               15
Figure 1: Publications relating to cloning in mammals (excluding mice), birds and fish




Cloning hit the headlines through work performed at Roslin Institute. The first paper
described the generation of ‘Megan and Morag’ from foetal cells and was published in 1996
(Campbell et al. 1996); the following year ‘Dolly’ was presented to the world (Wilmut et al.
1996). There are publications concerning the idea of cloning prior to these, all except one for
work with cattle. Subsequent to these publications, the output as measured by scientific
publications rose steadily to peak at about 100 per year in 2002. The importance of this field
is reflected in the emergence of at least one scientific journal dedicated to animal cloning.
The publication level has remained stable over the following two years (2003-2004). This
may reflect the amount of funding in cloning across the world, which may have reached a
plateau. In the first 5 months of this year (2005) only 20 publications were identified and if
this rate is sustained over the full year then the total could be about 50. This would be a
substantial decrease on the previous 5 years. Whether this trend will appear and what it
reflects with respect to animal cloning research is not clear.
The following species have been cloned: sheep, pig, cow, goat, rabbit, horse, mule, cat, fish
and dog. By far the most research has been with cattle, resulting in approximately 50% of
scientific publications. The next most studied is the pig followed by sheep. Combined, these
three species account for 70% of scientific publications.
In addition a variety of rare or endangered breeds within these species have been cloned.
Two key publications are worth noting in this regard. The first in 1999 (Wells et al. 1998)
used cloning to preserve the last surviving cow of the Enderby Island cattle breed; the
second in 2001 (Loi et al. 2001) demonstrated that post mortem cells can be used to recover
an animal. Currently, there is considerable interest in the Far-East, particularly China, in this
application of cloning.
Only recently, since 2001, have reports on the (animal) product quality, animal welfare and
other safety aspects have started to be addressed. To-date, these publications account for
only 3% of the total.




                                               16
Figure 2: Geographical distribution of publications relating to cloning in mammals (excluding
mice), birds and fish




It is perhaps through analysis of the geographic distribution of scientific publications that the
most interesting trends appear. The USA and Far-East have each produced approximately
the same number of publications. We are of the opinion that the full extent of work in China
and other countries in the Far-East was under-represented in the databases we searched.
Therefore we believe that the majority of scientific publications concerning cloning are from
this part of the world. This geographical distribution is all the more dramatic if the publication
output of last year and this year are analysed. During 2004 and up to May of 2005, 50% of
the scientific publications have come for the Far-East. Furthermore, 20% of the total output
from the Far-East has appeared during this period. This implies that, although a late entry
into the cloning research field, the Far-East is now, by far, the major contributor.
Europe, although the initial leader in this field, now lags behind both the USA and the Far-
East. We see no reason why this trend will not continue; if Europe aims to be a major player
in cloning research then it will have to focus on specific aspects of this subject.
2.2.7 GM
We again identified just over 500 publications. Although similar in number to that identified
for cloning, the distribution of publications concerning GM is distinct.
Figure 3: Publications relating to GM in mammals (excluding mice), birds and fish




This field started earlier than cloning with the first report of GM livestock in 1985 (Hammer et
al. 1985). This initiated the research into GM animals and the number of publications rose




                                               17
over the following years to 45-50 per annum in 1997. This rate has been sustained since
then, although as with cloning research, a possible decrease may be happening this year.
GM sheep, pigs, cattle, goats, rabbits, chickens and fish have all been reported. The primary
species of interest has been the pig with 32% of publications. The second most reported GM
animal are fish (15%), e.g. tilapia, trout and salmon. Notably, in 2004 and up until May of
2005, the rate of publications in birds and fish has been maintained, indicating considerable
current interest in these animals.
The main use of GM has been to address the goal of generating animal donors for
xenotransplantation surgery (22% of scientific publications). This trend is continuing with
21% of 2004 to May 2005 publications concerning xenotransplantation. The second
application is pharming (17%), however this interest appears to be reducing (13% of 2004 to
May 2005 publications).
We are less clear on the geographical distribution since the databases from which the
information was obtained were limited in affiliation recording. Nevertheless it was clear that
the majority of research had been carried out in the USA and Europe; with Europe displaying
a clear lead over the rest of the world. As with cloning, the Far-East is a late entrant into this
area of research but fast catching up. For the future, Europe needs to provide the support, on
focussed specific aspects, if it wishes to enable its current lead position to be maintained.
Timing is of the essence if the emerging momentum of the Far-East is to be challenged. In
this regard, the USA has already started to consolidate its efforts, through nationally funded
facilities, to combat the international competition. In the biological sciences, Europe has not
pursued this approach.
Figure 4: Geographical distribution of publications relating to GM in mammals (excluding
mice), birds and fish




2.2.8 Cloning and GM
The first publication using cloning to produce a transgenic animal was in 1997(Schnieke et
al. 1997). In both papers, the work was performed by a UK-based company. Indeed the
majority of scientific publications using cloning to produce a GM animal have had commercial
financial input. This largely reflects the huge financial cost of combining these technologies
and the potential commercial benefits. Reflecting this, just less than 100 scientific
publications were identified by our search.
To-date transgenic cattle, pigs and sheep have been produced combining cloning and GM
methodology.



                                               18
Table 3: First reports of GM livestock produced using cloning
 Date     Species     Commercial/public                   Country      Topic
 1997     sheep       Commercial (PPL Therapeutics)       UK           pharming
 1998     cow         Academic     (University        of USA           methodology
                      Massachusetts)
 2002     pig         both commercial and academic USA                 xenotransplantation
                      (Immerge BioTherapeutics Inc.
                      plus Universities in US and
                      Korea; and PPL Therapeutics)


Most work combining cloning and GM is currently performed in the USA and the Far-East.
There is interest within Europe, specifically in Germany and Italy.
Without question, at the moment the combination of cloning and GM provides the most
refined and precise form of genetic manipulation. However, it remains inefficient and
therefore expensive. Any technology that provides either more efficient or cheaper
technology can be viewed as a competitor to cloning and GM. As with all scientific
breakthroughs it is hard to see where this competitor will come from.
2.3 Future vision
The technologies of cloning and GM have several applications. First and foremost they
provide powerful tools with which to investigate biology. Secondly they can delivery novel
biotechnologies aimed at wealth creation and advancement of society. In addition, they may
offer refined animal breeding applications for agriculture that are also of use in companion
animals. Finally they provide a focus for public debate into science and society.
The potential impact on society of the first two and last applications should not be
underestimated. These technologies can significantly advance our understanding of biology
(within 5 years), our ability to overcome disease and improve human health and animal
welfare (5-10 years), and provide a vehicle for public engagement in science. Indeed, the
application of cloning and GM in these areas is already happening.
2.3.1 ES cells
One key target for the future, and one that could overcome the current limitations of cloning
and GM, is the derivation of ES cells from livestock species. There has been considerable
activity in this area for many years, many claims have been made but as of yet no truly
robust livestock ES cells have been reported. Combining GM in ES cells, with or without
cloning could constitute a significant advancement and, therefore, should be a major goal for
the future (5-10 years). Given the expertise available within Europe across a range of
species it is possible that this advance could be lead by Europe.
Finally, and importantly, it should not be forgotten that research with animal ES cells and
cloning can be expected to inform on surgical use of stem cells in human medicine. Thus
cloning and GM can be viewed as underpinning important developments in human health.
2.3.2 What species and to what applications?
Research into cloning and GM has largely focussed on improving and refining the
technology. As such the choice of species has not always been of primary focus. With regard
to application of cloning, most research has been with cattle and pigs, even though the first
cloned mammal was a sheep.
With regard to cloning and GM applications two issues have predominated. First for research
into xenotransplantation the majority of effort has been on generating transgenic pigs, latterly
more and more through the use of cloning, and initially in research projects in mice. With


                                               19
regards to pharming and the generation of animal bioreactors, the majority of research has
addressed producing proteins of biomedical importance in milk. For this the mouse has been
extensively used as a model, with more applied projects in sheep, goats, pigs, cattle and
rabbits.
In birds, all projects have had a commercial aim given the difficulty and expense involved in
generating GM birds; all work has been with chickens. It is possible that the recent
development of lentiviral vectors may enable expansion into more basic scientific projects
(i.e. what does a gene do in a given tissue?) and different bird species.
Fish transgenesis can be used to study gene function and regulation, e.g. in species such as
the zebrafish and medaka, which are considered important model species to understand
biology, and to improve the traits of commercially important species such as salmon and
trout.
In the following discussion we have reflected what the scientific community considers to be
the most likely areas of research (from direct questioning of a panel of scientists in the field
of cloning and GM) rather than merely cataloguing of all that has been reported in the
literature. In addition, future effort will be applied to developing the tools and proof-of-concept
evaluations in these areas; not surprisingly since some will provide intellectual property the
details of these experiments have not been made available to this report, therefore we can
merely highlight the areas that we anticipated to be pursued.
i. Animal breeding for agriculture
Uses of cloning and GM in animal breeding to produce food products that are close to
commercialisation are reported in Section 2 of this report.
Cloned and GM animals that have an increased ability to overcome or even resist disease
may be achieved in future, e.g. animals unable to catch or transmit BSE. In this regard, cattle
less susceptible to mastitis have been reported that carry a lysostaphin-transgene delivered
using the cloning method. One area that is attractive excitement, but has yet to be
demonstrated, is the use of RNA-interference to destroy infectious pathogens. The
considerable effort currently being applied to this topic should enable an evaluation to be
achieved within the next 5 years. However, in order to generate animals that are able to
resist all form of mastitis multiple transgenes will be required. Progress in the development of
vectors capable of delivering multiple transgenes could present a major step improvement in
the technology (within 10-15 years if given enough support).
ii. Xenotransplantation
Within the scientific community this topic raises most interest. Europe has an interest in this
topic through work in Germany and Italy. It is hard to gauge whether this reflects true
optimism that it will be successful and provide the much needed increase in donor organs or
that it posses a huge technical challenge for science to overcome. Again there is a need for
multiple transgenes if this application is to be realistic.
There is also considerable optimism for a similar application, the use of cloned and GM
animals as models of human disease (within 5 years). The aim would be both inform on the
disease progress and enable refined intervention strategies be designed. Possible examples
include generating pigs with retinitis pigmentosa or melanoma and sheep with cystic fibrosis.
iii. Bioreactors
Uses of cloning and GM to produce pharmaceuticals that are close to commercialisation are
reported in Section 2 of this report.
iv. Underpinning stem cell therapy
One very important legacy of cloning is the relatively new thinking that the differentiation
state of a cell can be altered. This opens up the possibility of stem cell therapies for human



                                                20
disease. Combine this with GM technologies and there is the real possibility of refined
surgical intervention in diseases such as cancer.
Research into cloning and GM underpins this exciting image of future medicine. Research
into cloning and GM of animals therefore provides a valuable and powerful tool in the
biologists’ armour to fight disease. If successful, the impact of this avenue of research on
human health and through providing the base of commercial ventures and wealth creation is
huge.
2.4 Public versus private research
The majority of cloning and GM research relies on public funding. The biggest projects are
commercially supported or performed in the private sector. Within Europe most public
funding research is performed in the UK, Germany and France with some in Italy and
Hungary. A reasonable, albeit indirect, measurement of the extent of publication funding can
be gained from the relative geographical distribution of publications.
With regard to international commercial funding for cloning research and GM, the USA is the
leader with Australia, New Zealand and China also with a major interest. Within Europe,
France and Italy lead with regard to animal projects.
There is considerable commercial interest in cloning and GM. Some is overt, e.g. those
companies that are involved in a strong public relations exercise with regard to cloning,
companies based on animal bioreactors. Other commercial interests are less transparent
and many scientists have association with commercial ventures. The opportunities in cloning
and GM for wealth creation and improvement in human health and animal welfare should not
be underestimated. Structures will require to be established to allow the flourishing of
commercial entrepreneurism while providing a robust publicly funded, therefore publicly
accessible, research base. Europe, including some national initiatives, can provide this
environment; indeed the EU has a proven track record in enabling this environment to be
established through the various Framework funding programmes that involve the academic
and commercial communities. This exciting and in all likelihood lucrative area of
biotechnology could provide benefits for Europe.
2.5 Main drivers for research
There are three main drivers for scientists for research into cloning and GM.
i. Scientific acclaim
There are two aspects of this that need to be considered. First, biological research is driven
by the desire to tease apart biological mechanisms and processes. At the top of the interest
list, biologists seek understanding of how animals (primarily humans) develop from an egg to
a living, healthy adult. Cloning and GM provide the means to pursue this quest. Secondly, a
scientist’s career is based on the number, quality and impact of the scientific publications in
their name. Research into the high profile area of cloning and GM provide an attractive
vehicle to achieve high impact publications; the publication of ‘Dolly’ made Professor Ian
Wilmut a household name.
ii. Commercial gain
The promise of commercial exploitation of this technology provides the driver for financial
support of research. With respect to cloning, while the commercial interest expands the
possibility of return on investment in cloning, in parallel there is an ever-decreasing interest in
supporting academic development of the technology as it becomes more routine within
commercial companies. The same can not be said for GM, with or without cloning, were
there is an increasing need for, and therefore commercial support of, technology
development.
iii. The promise of providing solutions for human betterment.
Finally underlying most individuals, scientist or not, is the desire for human betterment.


                                                21
SECTION 3: OVERVIEW OF COMMERCIALISATION ACTIVITIES WORLDWIDE
3.1 Introduction
This section aggregates original data and publicly available information of commercial
activities worldwide in animal cloning and/or genetic modification. The information has been
collated and presented in the form of a potential pipeline of products expected to arrive in the
market in the next 5 years, next 5-10 years and after 10 years.
Application of GM, cloning and both GM and cloning technologies to the following sectors are
considered: food production, molecular pharming, xenotransplantation, pets, sporting
animals and endangered species.
3.2 Methodology
Original data for this study were collected by interviews and questionnaire.
Questionnaires were sent to approximately 50 organisations, only three were returned
completed despite repeated prompts. The questionnaire is reproduced in Appendix 3. The
basis for selection of the interviewees was: to cover the range of application sectors, provide
geographical spread, cover the technology combinations included in the survey and the
ability of the interviewees to give a balanced, realistic analysis of the prospects for
application of these technologies to these sectors. Interviewees were based in USA, Canada,
Australia, Argentina, France and UK. Twenty six interviews were requested and 15 were
successfully completed. Three of these interviews were face-to-face the rest were telephone
interviews. Three of those interviewed work in senior management or research positions in
public institutions the rest hold a range of senior positions in private organisations, including
research directors or CEOs. Overall, the level of co-operation in the survey was not good,
reflecting a similar experience with a previous EC research project ‘Mammalian Cloning in
Europe: Prospects and Public Policy” (Martin et al., 2000). In particular, we experienced
significant difficulty collecting information on commercialisation activity in the Far East. To
assist in understanding publicly available information in the Far East, native speakers of
Japanese, Korean and Chinese were employed to survey the information available from
these countries via the Internet.
Several factors may have influenced this low response rate:
   reluctance to divulge commercially-sensitive information
   a number of companies we identified either did not exist any more or had divested their
    cloning/GM activities
   several companies were collaborating on the same project and have been counted as
    separate companies although only one of them is commercializing the technology and
    therefore able to answer the questions we posed;
   the EU is considered a low priority for a number of the companies commercializing this
    technology;
   the controversial nature of the work engendering a general caution and apprehension
    about revealing any information about the activities for fear of bad publicity even though
    assurances about confidentiality were given and general uncertainty about the motives
    behind the survey.
Therefore, most of the information in this report was collected from a survey of literature,
websites and other publicly available sources. This is a rapidly developing area of technology
and every effort has been made to ensure that the information presented is accurate.
However, it should be stressed that this study is very much a snapshot in time. Many of the
companies involved in GM and cloning developments in animals are small companies and
they operate in an area of significant commercial and regulatory risk.




                                               22
The proposed pipeline of potential products is based on the publicly available information on
the plans of the companies concerned, responses during interviews, reported positions of
relevant regulators and the opinions of the survey team in the light of the collated
information. This is therefore necessarily a ‘best-guess’ based on the information available.
Since significant weight was given to the views of those directly involved in the development
the pipeline presented is more likely to be optimistic rather than pessimistic.
3.3 Inventory of the companies active in the GM and/or cloned animal sector
Based on a survey of web sites and literature plus 15 interviews and 3 questionnaires we
found there are 35 companies worldwide involved in producing GM animals, cloned animals
or both GM and cloned animals (parent companies are listed below but not included in the
total). Of these, 63% are in N. America, 14% in Europe and 11% in Asia ( the figure for Asia
may be an underestimate since it was very difficult to obtain information). By sector the
distribution of companies is food production 15%, pharming 40%, xenotransplantation 10%,
pet 12.5%, sporting 5%, endangered 10% and other 7.5%, 5 companies are active in more
than one sector.
Only products from GM, GM and cloned or cloned animals are considered in Table 4.
Several of the companies have a portfolio of other products. A start-up was defined as any
small company established from 2001, although it was not possible to determine when some
companies formed.
Table 4: Inventory of companies active in the GM and/or cloned animal sector
 Sector     Company         Activity   Product       Product      Product in Start-up   Geograp
                                       on market     awaiting     developme SME         hy
                                                     regulatory   nt         Large      (based
                                                     approval                           on R&D)
 Food       Viagen/Exet C              Show          Cloned                    SME      USA
 producti   er      Life               cattle        pig semen
 on         Sciences/Pr
            olinia
            A/F Protein GM                           GM           GM fish      SME      Canada
            Canada                                   salmon
            Inc./AQUA
            Bounty
            Technologie
            s Inc
            Celentis        C                                     Cloned       ?        Australia
            Ltd/Clone                                             beef and              /New
            International                                         dairy cattle          Zealand
            /
            Cyagra          C                                     Cloned       SME      USA
                                                                  beef and
                                                                  dairy cattle
            Yangling        C          ?             ?            ?            ?        China
            Keyuan
            Cloning Ltd.
            MaRS            GM                       GM pig                    SME      Canada
            Landing
 Molecul    AquaGene        GM                                    Factor VII SME        USA
 ar         LLC                                                   in tilapia



                                                23
Sector    Company       Activity   Product     Product      Product in Start-up   Geograp
                                   on market   awaiting     developme SME         hy
                                               regulatory   nt         Large      (based
                                               approval                           on R&D)
pharmin   AviGenics     GM                                  alpha         SME     USA
g         Inc                                               interferon
                                                            in chickens
          Bio    Sidus GM+C                                 Growth     SME        Argentin
          S.A.                                              hormone in            a
                                                            cattle
          Bioprotein    GM/GM                               Proteins    SME       France
          Technologie   +C                                  and
          s                                                 vaccines in
                                                            rabbits
          GeneWorks     GM                                  Proteins in SME       USA
                                                            chickens
          GTC           GM                     Antithrom                  SME     USA
          Biotherapeu                          bin III in
          tics/                                goats
          Genzyme
          Transgenics
          Hematech/K GM/GM                                  Mono and SME/Lar      US/Japa
          irin brewery +C                                   polyclonal   ge       n
          Company                                           antibodies
                                                            in    cattle
                                                            and mice
          Abgenix       GM/GM                               Mono and Large        Canada
                        +C                                  polyclonal
                                                            antibodies
                                                            in mice
          Origen        GM                                  polyclonal    SME     USA
          Therapeutic                                       antibodies
          s                                                 in chickens
          Pharming      GM+C                                C1           SME      Netherla
          NV/ ProBio                                        inhibitor in          nds
                                                            cattle
          TranXenoG     GM                                  Protein in SME        USA
          en                                                chicken
          Viragen       GM                                  Proteins      Large   UK
                                                            and
                                                            antibodies
                                                            in chickens
          CIMAB S.A     GM                                  Recombin Large        Cuba
                                                            ant
                                                            proteins in
                                                            animal cell
                                                            lines




                                         24
Sector     Company       Activity   Product        Product      Product in Start-up      Geograp
                                    on market      awaiting     developme SME            hy
                                                   regulatory   nt         Large         (based
                                                   approval                              on R&D)
           Vivalis       GM                                     Recombin SME             France
                                                                ant
                                                                proteins in
                                                                animal cell
                                                                lines
           BioAgri                                              Proteins in ?            USA
                                                                chickens
Xeno       MGen Bio      GM+C                                   Pig           Start-up   Korea
                                                                pancreatic
                                                                islet cells
           Alexion       GM+C                                   Pig organs    ?          USA
           Pharmaceut
           icals Inc
           Revivicor     GM+C                                   Pig organs    SME        USA
           Ximerex       GM                                     Pig organs    ?          USA
Pet        Allerca       GM                                     Pet cats      ?          USA
           Genetic     C            Pet cats                                  SME        USA
           Savings and
           Clone
           PerPETuate    C                                      Cloned        SME        USA
                                                                pets
           Yorktown      GM         GM                                        ?          USA
           Technologie              zebrafish
           s
           TaiKong/Az    GM         GM                                        Large      China/T
           oo                       zebrafish                                            aiwan
Sporting   Viagen/Exet C            Rodeo                                     SME        USA
animals    er      Life             bulls
           Sciences/Pr
           olinia
           Cryozootec    C          racehorse                                 ?          France
           h                        s
Endang     Cyagra        C          ?              ?                          SME        USA
ered
           Cryozootec    C          ?              ?            ?             ?          France
species
           h
           EMBRAPA       C          Junqueira                                            Brazil
                                    cattle
           Genetic     C            African                                   SME        Brazil
           Savings and              wildcat
           Clone
Other?     Evergen       C                                      ?             ?          USA



                                              25
 Sector     Company         Activity    Product      Product       Product in Start-up       Geograp
                                        on market    awaiting      developme SME             hy
                                                     regulatory    nt         Large          (based
                                                     approval                                on R&D)
            Biotechnolo
            gies Inc (all
            of       the
            above)
            PharmAthen ?                                           Butyrylchol   Start-up    USA
            e                                                      inesterase
            (bioterroris
            m)
            MaRS            GM                       GM pig                      SME         Canada
            Landing
            (environmen
            tal impact)


3.4 Summary of products in the pipeline
3.4.1 Food production
i. Products already on the market
In the strict sense, consumers across the world are already eating meat and milk from cloned
cattle. These are the naturally occurring clones that arise from monozygotic twin births. Twin
births in the cattle are in the order of 2% of pregnancies (Fricke and Shaver, 2004) and it has
been estimated that 10% of twin births are monozygotic (Chris Haley, personal
communication), therefore, in the order of 0.2% of beef and milk is from naturally occurring
clones. Monozygotic births are also believed to be a rare event in pigs.
There are also clones that arise from the practice of embryo splitting when in vitro fertilization
(IVF) is used for the dissemination of high merit genetic material in cattle. The process began
in the early 1970s as a way of improving the yield of embryos and the likelihood of
establishing a pregnancy, and gained wide acceptance in commercial use (Seamark, 2003).
Chronologically, the next technical development was the ability to clone embryos by using
undifferentiated cells from developing blastocysts or, later, from cultured embryonic stem
cells, through nuclear transfer. Using cells from blastocysts, it was also possible to undertake
serial cloning, making clones of clones in vitro before implantation in the recipient dam.
It has been reported that these techniques have been widely adopted with over 2000 calves
derived from embryo-splitting and over 200 derived from embryo nuclear transfer registered
with the US Holstein Breeders Association by October 2002 (Norman et al., 2004). As far as
can be ascertained there were no regulatory constraints on these animals and some of them
will have entered the food chain. Anecdotally the popularity of these techniques has lessened
although embryo transfer is still used especially for export of embryos between countries.
Despite the natural occurrence of clones and clones derived from embryology techniques,
there are many that think cloning was not a reality until the success of adult somatic cell
nuclear transfer (SCNT), or adult cloning. Technically, the 200 calves produced from
embryonic cloning are also produced from somatic cells, so they are also SCNT clones and
they may have entered the market in the United States.
Whilst we were unable to verify any ‘approved’ commercial use of adult SCNT cloned or GM
livestock in the food sector anywhere in the world, there are growing numbers of cloned
livestock in private hands, especially in the USA. The use of such clones in food production


                                               26
is not currently illegal in the USA, but is subject to a voluntary moratorium, issued by Centre
for Veterinary Medicine (CVM) July 13th 2001, until FDA guidance is available. A small
number of show or elite animals have been cloned by SCNT and are in the hands of private
farmers/breeders. Larger numbers are in the hands of research organisations or commercial
companies developing the technologies. Worldwide the number of cattle, pigs, sheep and
goats produced by SCNT cloning must now be into the thousands of animals, though many
of these animals will no longer be alive.
There are also significant and growing numbers of GM or cloned and GM animals in
research populations in various countries.
Aside from a very small number of identified instances of accidental entry into the food chain
(e.g. University staff taking meat home or animals going to rendering rather than incineration)
there is no indication that any of these GM or adult SCNT cloned animals have or will enter
the human food chain, but the possibility of small scale inadvertent (or deliberate) entry to the
food chain exists.
Much more likely in a short timescale is the entry into the food chain of meat or milk from the
progeny of SCNT clones.
In a recent press article, a US cattle breeder claims to have sold more than 500 units of
semen from five male clones of a prize bull called Full Flush. The same article indicates that
no-one knows for certain whether any of the meat from the progeny of these cloned bulls has
entered the human food chain (Roosevelt, 2005). A Swiss company has also recently been
reported to have imported semen from a US bull that is the offspring of a clone and used it to
inseminate 300 cows (Swissinfo, 2005).
ii. Products that may be on the market within 5 years
GM livestock
Fast-growing GM fish (Salmon) have been developed in N. America that are awaiting
regulatory approval, principally for direct sale to fish farming markets in N. America, Asia and
S. America. In the opinion of the developers, the structure and influence of the large retail
supermarkets in the EU, rather than direct consumer acceptance is considered a specific
barrier to uptake of this technology in the EU. Communication with regulators and production
of credible risk assessments forms an integral part of the commercialisation process with
both food safety and environmental safety being addressed. The benefits of this kind of
technology are described as cheaper and more environmentally-friendly source of omega-3
rich fish. It is possible that approval for production of GM fish may be granted within five
years. At least eight species of fish have been genetically modified for growth enhancement;
these include grass carp, tilapia, rainbow trout and catfish (WHO, 2005).
GM pigs developed at the University of Guelph in Canada with a novel digestive enzyme
(phytase) were expected (by the developers) to be on the market within the next 5 years, but
not necessarily in the EU. Reduction of the environmental impact of phosphate from pig
production is the key benefit of these animals. Numerous challenges to commercialisation
were described by the developers including; lack of written regulations, consumer
acceptance and the time required to incorporate the technology into existing breeding
schemes. The rise of regulation on environmental impact of livestock production was
expected to increase demand for these GM animals. The priority markets for GM phytase
pigs are North America, SE Asia, S. America and Russia.
For terrestrial livestock, the general view expressed in our survey was that GM livestock
would not be in commercial use before 2010, though some commentators, especially the
developers of the products mentioned above, thought this possible.
Cloned livestock




                                               27
There is a widely held view among those well informed about the technology landscape, that
cloned livestock (especially pigs and cattle) will be used within the food chain somewhere in
the world before 2010. The expectation is that within this timescale, it would not be economic
for cloned animals to be used directly for food or milk production, but that clones would be
used as parent sires of slaughter generation pigs, beef cattle and possibly also milk-
producing dairy cows. The main argument for the use of such cloned sires is economic. As
one interviewee put it “If you save 1-2% of costs over millions of pigs the return [on
investment] is certainly there.”
Cloned parent males
The pig industry widely uses artificial insemination (AI) and the norm is for these AI sires to
be of high genetic merit. However, the number of inseminations possible from any one male
is limited and the supply of the highest merit animals produced from breed improvement
programmes is also limited. The number of high merit animals originating from the top of
breeding programmes is simply too few to meet the need for AI sires to be used across the
commercial sow population to produce slaughter generation pigs. For example, in pig
production, AI sires might typically be from the top 10% of all performance-tested males in
terms of overall economic merit for efficiency of production. Using clones, it should be
possible to ensure that all the males used in pig AI are of the highest merit, e.g. top 1% or
top 0.5%. Taking into account the fact that cloned boars would be genetically older, our own
calculations (assuming one generation of genetic lag) show that there could be significant
economic benefits from the use of cloned AI sires in pig production (see Appendix 2).
Conservatively estimated, these benefits to the farmer are in the order of €1.2-1.5 per pig,
equivalent to at least €145m per year across the EU if the technology were to be used for
50% of production. This estimate does not take into account downstream benefits for
example improvements in processing efficiency. There are no technological barriers for the
uptake of AI technology. The very smallest farmers maybe less likely to take up AI, because
their infrequent use of this technology may result in a low proficiency.
The above calculations together with comments from those interviewed indicated that for
pigs, the cost of a cloned animal would need to be less that $10,000 (€8,000) for the
economic case to be viable. All indications are that the actual costs charged by service
providers will be less than this, probably below $5,000 (€4,000) when volumes are high
enough. Pig breeding companies maintain commercial confidentiality over the prices attained
for boars sold to AI studs, but considering the value of the boar to the AI stud in terms of
inseminations, a realistic value would be in the order of €2000 to €3000 per boar. This
means that a cloned boar is likely to cost little more than twice the cost of a conventional
boar, assuming of course, that the high merit animals to be cloned do not increase markedly
in value if cloning became commonplace.
One cloning company sees the pig industry as its first high volume market and estimates the
market potential to be a few thousand cloned boars per year within a few years of approval.
This suggests a market size within the US into tens of millions of dollars per year. These are
probably realistic aspirations. Our own calculations estimate the number of boars in AI studs
to be in the order of 38,000 and 14,000 in the EU-25 and US respectively. Assuming these
boars are replaced at 50% per year, the EU market for cloned sires to AI studs probably has
an upper value in the order of €75m per year. Globally the market could be over €250m per
year.
In the case of dairy bulls, there is less clarity about economic prospects within the next five
years. Artificial insemination is also widely used in the dairy industry, but the AI technique
used generally means that a single bull can be used widely without the supply of semen
becoming limiting. However, by the time that a bull is known to be of high genetic merit, he
might be 5 or 6 years old. There have been cases of high merit animals dying or being
injured and euthenized long before their potential productive life has expired. Discussion with
dairy breeders and AI operators indicated that one use of cloning might be insurance against



                                              28
the loss of individual animals of high merit. It would not be necessary to produce the clone as
insurance, merely to store somatic cell lines, such that the animal could be cloned after its
death.
Viagen would put dairy bulls third on their list of expected market development after first pigs
and then beef cattle. A US business called Infigen had placed significant emphasis on dairy
animals before the business was closed down. Research in Australasia by AgResearch in
association with Clone International has focused on the dairy bull. The clone International
website lists the price of cloning a bull at AU$50,000 (just over €30,000). We understand that
the market potential seen by these collaborators is not necessarily for their own Australasian
market, but more the sale of cloned elite dairy bulls to other countries, especially Asia. Clone
International has a non-exclusive license from Geron Corporation (owners of the ‘Dolly’
somatic cell nuclear transfer technology) to operate in the Peoples Republic of China and
exclusive licenses for Australia and New Zealand. The sale price for cloned elite dairy bulls
was estimated by one interviewee as AU$250,000 (€155,000). Another cloning company
Cyagra, stated in 2002 that they charged €15,570 for a cloned calf.
The ability to clone animals from cells taken from an animal soon after slaughter provides a
particular potential advantage for the beef sector (and to a lesser extent perhaps the pig
sector). Breed improvement programmes for beef cattle are based on relatively small
populations of a few thousand tested animals per year and progress in the rate of breed
improvement is limited by, amongst other things, the available population size.
Most commercial male cattle further down the production pyramid are produced as castrates
and even if extreme individual males were identified to be superior in any way, they would
not be available for breeding. However, cells taken from a castrated male beef animal will, of
course, produce an entire male animal when used for SCNT cloning.
Carcase quality traits are very important in beef production and are highly heritable (a
relatively high proportion of the variation seen is due to genetics rather than environment).
More than one interviewed organisation proposed that it would be economically beneficial to
identify extreme high merit animals (for carcase quality traits) at slaughter and clone entire
male cattle from cells of the carcase. This animal could perhaps be the best in 10,000 or
more animals rather than the animal estimated to be the best from a smaller population of
live purebred animals.
It would even be possible to make sure that the carcase selected for cloning was produced in
the environment in which its progeny would be produced, thus ensuring the best possible
match of genes and environment. However, there are significant risks in this approach: i)
whilst it is known that carcase traits are highly heritable it is not clear what proportion of a
given cloned animal’s merit in carcase traits will be passed on to progeny, and ii) even if the
carcase traits of progeny of the clone are indeed superior, overall economic merit depends
on a broader range of traits, such as fertility and growth efficiency. It may not be possible to
have confidence in the overall merit of any particular cloned beef bull until he has significant
numbers of progeny. Some limited research on this approach is underway in New Zealand.
The studies needed to determine the potential for this approach are relatively simple, but not
small scale. Until there is clarity on the ability to sell the progeny of clones into the food
chain, such large scale studies are unlikely to be undertaken by industry.
Some of the same arguments about selecting the very best individuals from the slaughter
generation may also apply in pigs. One example, cited by interviewees, was the potential to
select pigs of the very best meat quality (as can only be assessed at slaughter) for cloning to
produce very high quality pig meat for those markets that pay premium prices for high quality
e.g. Japan.
Sheep have been cloned in New Zealand for research purposes. Rare individuals from
breeding lines selected for parasite resistance/susceptibility were cloned to increase the
number of sires thought to be segregating the genes responsible for variation in resistance. It



                                              29
has been claimed that a Chinese cloning company which has successfully cloned a goat has
received 2 billion yuan (€0.2 billion) worth of orders from 14 provinces and cities for cloned
sheep and cattle (Chan, 2001). However from another source we have been informed that
China does not have any companies commercializing cloned or GM animals.
iii. Use of cloning in breed improvement
It is generally considered that cloning has little place in breed improvement programmes. By
definition clones produce animals of the same rather than better genetic merit. Breed
improvement is a continuous process with better animals produced in each generation. This
means that the ‘best’ animal for a given environment, that might be cloned, will be
superseded by a ‘better’ animal within a short timescale. Cloning may provide a multiplication
step downstream of breeding programmes, speeding up the dissemination of the best
combinations of genes, but the animals to be cloned would likely change each generation.
There may be an exception to the generalisation that clones have little value in breed
improvement. Some traits such as meat quality are very difficult to predict on live animals
available for selection, and where breeders wish to select for meat quality, they currently
need either or both information from measurements on dead relatives of the animals
available for selection, or genetic markers that predict a useful proportion of meat quality.
Phenotypic selection for meat quality using information from relatives is expensive and rarely
utilised. Some genetic markers for meat quality are available, but they explain a relatively
small proportion of the genetic variation in quality traits.
The exception comes from the previously described potential to produce clones from the
carcases of recently slaughtered animals. Where breeders wish to select for improvement of
carcase traits, it may make sense for some clones from dead animals to be utilised at the top
of the breed improvement pyramid.
SCNT cloning opens up a new option of accurately measuring meat quality on carcases and
selecting cells from the best quality meat for cloning. This approach could be used to
increase substantially the population size available for selection (because high merit animals
for some quality traits may originate from the slaughter generation, though they are likely to
be lower merit for some other traits), whilst also enhancing the accuracy of selection for
difficult to measure traits.
One interviewee described their own calculations relating to meat quality in pigs. They were
not prepared to disclose the calculations, but indicated that there was real potential to use
clones of dead pigs to enhance their capability to improve meat quality though selection. The
basis of their calculations was to identify small numbers of carcases from male pigs of
exceptional meat quality on the slaughter line and to produce clones from these carcases.
These males would then be used at the top of the breeding pyramid to improve the genetic
merit (for meat quality) of a breed or line selected for producing animals for markets where
there is a premium for meat quality. If clones were to be used in this way, some meat from
the surplus purebred progeny of the clones may well enter the food chain, but it could be two
or more generations down the breeding pyramid that (great-) grand progeny of the clones
would be the food-producing animals.
A potential competing technology for selection on difficult to measure traits could be the use
of cloning through embryo splitting to produce two animals of identical genotype. This would
enable one animal to be extensively tested (including post slaughter measurements) whilst
its live clone was retained for potential breeding use. This approach would effectively double
the number of animals at the top of the breeding pyramid (though only one of each pair
would need to be performance tested) and is unlikely to be financially viable.
It should be noted that the potential benefits of the use of cloning in livestock production are
not all economic, higher performance animals will likely be more efficient users of inputs and
will produce less pollution and waste per unit output, potentially generating significant
environmental benefits.


                                              30
iv. Products that may be on the market beyond 2010
GM livestock
Whilst there was general scepticism that GM terrestrial livestock would be in the food chain
within five years, there was more optimism that some of the ‘better’ GM applications would
be in the market within ten years and even greater optimism beyond this timescale. As one
interviewee put it “I feel we will one day look back and wonder what all the fuss was about”.
There was a general consensus, perhaps not surprising given the experience with GM
plants, that a key driver to uptake of GM livestock would be consumer acceptability and that
the first applications to progress to market should be those with clear benefits to consumers,
such as enhanced nutritional quality. Applications that had benefits for the livestock (e.g.
disease resistance) were also considered more likely to be acceptable than applications that
were perceived to be about production efficiency.
A number of research studies on GM livestock have already been completed. Notable
examples are the various studies on animals with extra copies of the growth hormone gene.
As far as we can determine, none of these animals are progressing towards commercial
application with the exception of the GM salmon.
Food applications that were identified during the survey as topics of current research
included:
   Changing the nutritional composition of milk and meat
   Improving resistance to viral disease
   BSE resistant cattle through PrP knockout
   Changed milk functionality in dairy cows (casein genes)
   Cows with antimicrobial compounds in milk for reduced mastitis
   Pigs with changed milk production
   Pigs with novel digestive enzymes
   Altered sex ratios in poultry
   Increased breast muscle weight in broiler chickens
   Fish with lysozyme from other species as an antimicrobial
   Induced sterility in prawns to facilitate trade in improved prawn varieties that cannot then
    be used as broodstock by customers
   Sex linked muscular hypertrophy (mysostatin deletion) in cattle
   Modified rumen flora bacteria for protection against plant toxins
   Meat and milk from cloned animals
Those developing cloning for the dairy industry have long held out the prospect of milk
production from herds of cloned dairy cows. The main potential benefits are perceived to be
economic, with cloning opening up the possibility that the highest genetic merit female
animals can be multiplied and sold as embryos to dairy farmers. In this context highest merit
is often taken to be the best yielding cows, but there is no reason they should not be the best
cows in terms of overall merit; taking account of the need to address potential welfare
problems in diary cows such as lameness or reduced fertility.
Cloning also opens up the possibility of increasing the potential to exploit the advantages of
heterosis by using crossbred females in milk production. Overall efficiency of milk and meat
production could be improved if dairy cows were implanted with either female embryos of
cloned high merit cows (purebred or crossbred) to replace the females, or male embryos of


                                               31
(cloned) high merit beef cattle for beef production. Such a process would eliminate the
production of dairy breed males that are of very low value and are often slaughtered soon
after birth. However, there are significant problems to overcome before this could be a
reality, with perhaps the greatest problem being the Large Offspring Syndrome seen with
some embryo transfer calves.
The use of herds of cloned dairy cows offers one further potential benefit. At the moment the
environment provided for any cow (e.g. feed composition and management routines) are
aimed at the average cow. Herds of cloned cows should enable management routines to be
optimised to the genotype of the cow. However, there are also additional risks, the main one
being the absence of genetic variation within the herd. This could mean that 100% of the
herd may be affected by any new disease entering the herd.
Published calculations Faber, D. C., et al. (2004) suggest that the potential economic return
from cloned dairy cows in the United States might be approaching $1000 (€800) per cow
over her lifetime. Clearly this is significantly less than the current cost of cloning and
commentators suggest that the use of SCNT cloned dairy cows is unlikely to be taken up
widely until the cost per cloned embryo can be offered at as little as $50 (€40) (Seamark,
2003).
The first commercially available GM food products of animal origin are likely to be GM fish
which may become available within the next 5 years (subject to resolution of regulatory and
other issues). Subsequently food products from cloned and/or GM pigs and cattle may also
become commercially available, as shown in Figure 5. Table 5 gives more detailed
information on the potential future food products.




                                             32
Figure 5: GM and cloned animals for food production in the pipeline for three different time
periods



   Period 2005-2010
   Group    1:   next   5
   years
                                 Period 2010-2015
   Semen, offspring, fish
   meat and derivatives          Group 2: next 5 to 10yrs
   from growth hormone
   GM salmon                     Semen, offspring, fish meat and         Period after 2015
                                 derivatives    from      growth
   Semen, offspring, fish                                                Group 3: more than 10
                                 hormone GM carp
   meat and derivatives                                                  yrs
   from growth hormone           Semen, offspring, fish meat and
   GM trout                      derivatives    from      growth         GM livestock with
                                 hormone GM tilapia                      disease resistance
   Semen, offspring, from                                                traits
   cloned cattle                 Semen, offspring, fish meat and
                                 derivatives    from      growth         GM livestock with
   Semen, offspring from         hormone GM catfish                      altered meat and
   cloned pigs                                                           milk composition
                                 Semen, offspring, GM pigs, pig
                                 meat and derivatives from GM            GM livestock with
                                 pigs                                    altered reproductive
                                                                         traits e.g. sterility
                                 Cloned cattle and milk, beef
                                 and derivatives from cloned
                                 cattle
                                 Cloned pigs, pig meat and
                                 derivatives from cloned pigs




Table 5: Description of GM and/or cloned animals for food production
 Description of GM and/or cloned animals              Product
 A GM Yorkshire breed of pig producing the Semen straws, pigmeat, pigmeat
 bacterial enzyme phytase in the salivary glands products, and other derivatives of pigs
 and hence using plant phosphorus more efficiently.
 GM Atlantic salmon and trout with enhanced Semen straws, eggs, fish fishmeat,
 growth rates. Salmon carry Chinook salmon growth fishmeat products, and other
 hormone driven by promoter for antifreeze derivatives of fish
 production in ocean pout.
 GM trout, carp, tilapia and catfish with enhanced
 growth rates.
 Progeny of cloned elite boars                        Semen straws, pigmeat, pigmeat
                                                      products, and other derivatives of pigs
 Progeny of cloned elite bulls of beef breeds of      Semen straws, beef and beef
 cattle                                               products, leather and other derivatives
                                                      of beef production



                                             33
 Description of GM and/or cloned animals               Product
 Progeny of cloned elite bulls of dairy breeds of      Semen straws milk and milk products,
 cattle                                                beef and beef products and leather at
                                                       slaughter
 Cloned dairy cows (probably small numbers until Semen straws, milk and milk
 costs fall dramatically)                        products, beef and beef products and
                                                 leather at slaughter


3.4.2 Molecular pharming
Pipeline products are described in figure 6.
Figure 6: GM and cloned animals for pharmaceutical production in the pipeline for three
different time periods




     Period 2005-2010
     Group 1: next 5 years

     human Growth hormone
     from GM+cloned cattle             Period 2010-2015
     Antibodies           from         Group 2: next 5 to 10yrs
     GM(+cloned?) rabbits
     human anti-thrombin       III     human albumin       from     Period after 2015
     from GM goats                     GM+cloned cattle
                                                                    Group 3: more than 10 yrs
     human        c1   esterase        human alpha-1 anti           human      monoclonal
     inhibitor from GM goats           trypsin from GM+cloned       antibodies       from
                                       cattle                       GM+cloned cattle
     human interferon alpha-
     2b from GM chickens               human   insulin     from     human       polyclonal
                                       GM+cloned cattle             antibodies        from
     human fibrinogen        from
     GM cattle                                                      GM+cloned cattle
     human lactoferrin       from                                   protein products     from
     GM cattle                                                      GM poultry
     human collagen from GM
     cattle


Biopharm animals are genetically modified to include one (or more) protein coding genes
from another species. These proteins can be extracted and used as drugs, biologicals, food
additives or other products of commercial value for human or veterinary use. The substance
is harvested and purified from the milk, blood, eggs or other tissue of the GM animal. The
genetic modification can be 1) germ line, heritable modification or 2) somatic cell or 3) gene
therapy. The animals act as “bioreactors” to manufacture large quantities of a protein.
Harnessing the metabolic capabilities of animals to produce proteins has advantages over
various other methods that are currently used for industrial production of proteins.
Animal cell line fermenters have limited capacity, bacterial systems have large capacities but
are limited to the production of simple non-glycosylated proteins and cannot achieve the



                                               34
post-translational modifications to produce functional human proteins. Animal cell culture
produces glycosylated proteins but at low yields.
Fungal systems allow efficient production of some secreted proteins, however addition of
mannose sugar residues during glycosylation can alter the properties of the protein. Addition
of different sugar residues is also a problem when using plants as bioreactors. Plant
bioreactors have the advantages that they do not carry pathogens that may be harmful for
human health and they do not contain any similar proteins reducing the difficulties associated
with producing and separating pharmacologically active proteins. Plants are emerging as a
promising alternative to conventional platforms for the large-scale production of recombinant
proteins. This field of research is developing rapidly and several plant-derived recombinant
proteins are already in advanced clinical trials. However, the full potential of molecular
farming can only be realized if we gain a fundamental understanding of biological processes
regulating the production and accumulation of functional recombinant proteins in plants to
ensure appropriate levels of protein production and consistency of product quality. Recent
studies indicate that species- and tissue-specific factors as well as plant physiology can have
a significant impact on the amount and quality of the recombinant product. It is expected that
advances to overcome limitations and/or selection of appropriate species will be possible
within 5-10 years (Fischer et al. 2004).
Baculovirus systems can produce a wide range of proteins but have yet to be scaled up to
industrial levels.
Two principal methods are favoured for large-scale commercial production of recombinant
proteins in GM animals, extraction from; i) milk of cattle, sheep, goats or rabbits or ii) chicken
eggs.
The mammary gland has evolved to produce large volumes of protein giving milk numerous
advantages as a source of recombinant human protein. Proteins can be obtained from
serum, but it is difficult to separate recombinant proteins from endogenous proteins, many
proteins are unstable in blood and sufficient quantities of proteins cannot be harvested from
serum. Some human proteins are bioactive across species and when expressed
transgenically in other mammals may result in deleterious health effects to the animal.
Several human recombinant therapeutic proteins are being produced in the milk of GM and
cloned cattle and goats and are at various stages of clinical trials. One of those we
interviewed believed there to be 50-100 “blockbuster applications” for GM and cloning where
pharmaceutical products will be produced in completely contained “pharm” facilities. No
products from GM animals have been approved for sale but several are in the final stages of
clinical trials and therefore relatively close to market.
At least one company is using GM rabbits to produce human therapeutic proteins in rabbit
milk, these proteins are currently progressing through clinical trials. Rabbits are genetically
closer to humans than any other dairy animals, have short generation time and are cheaper
to produce and maintain that cattle or goats. In addition rabbits have been successfully
cloned, but we could not find any evidence of cloning being directly applied to rabbits to
produce pharmaceutical products.
An alternative system to milk for manufacturing large quantities of human recombinant
proteins is the chicken egg. The typical chicken egg white contains 3-4g of protein, more
than half of which comes from a single gene, ovalbumin. Using the ovalbumin gene to
express a recombinant protein could potentially yield up to a gram of recombinant protein per
egg. On average a hen lays 252 eggs per year. Several human vaccines are currently
manufactured in chicken eggs.
Chicken eggs have several advantages as a system for manufacturing human recombinant
proteins. The evolutionary distance between birds and mammals means that chickens do not
usually recognize mammalian proteins, although they have similar post-translation processes



                                               35
of glycosylation and phosphorylation. Chickens also have a considerably faster generation
time than goats or cows.
The above advantages of chickens as bioreactors also apply to using GM fish to
manufacture human recombinant therapeutic proteins. There is one example of fish being
used as bioreactors where GM tilapia have been produced that express human coagulation
factor VII in their blood.
Table 6 compares average annual milk/egg yield, yield of recombinant protein per litre of milk
or egg and total annual yield of recombinant protein between cow, goat, sheep, rabbit and
chicken.
Table 6: Annual yields of recombinant protein from various sources
 Animal         Average annual milk/egg Yield of recombinant Total         yield               of
                yield (litres/ no.)     protein/litre or egg (g) recombinant
                                                                 protein/year (g)
 Cow            7,000                         2g                         14000
 Goat           850                           4g                         3400
 Sheep          450                           4g                         1800
 Rabbit         15                            5g                         75
 Chicken        252 eggs                      1g                         252


Generation intervals for these animals vary considerably, cow 140 weeks, goat 64 weeks,
sheep 52 weeks, rabbit 18 weeks, chicken 18 weeks.
To choose the animal system to produce each recombinant human protein in GM animals
the protein yield required and cross-species bioactivity would have to be assessed on a case
by case basis.
Using GM animals to produce therapeutic proteins is described as economically viable.
Transgenic protein production costs are low and production volumes high making the cost of
producing a protein transgenically approximately one-tenth that of standard cell culture
production (Jain 2004). High protein expression levels in GM livestock, concentrated raw
material, and efficiency in purification make the economics of “pharming” in animals highly
cost-effective. The ultimate cost of transgenic proteins is significantly lower than those
produced conventionally, reducing the cost of treatment using transgenic sourced product
considerably.
An industry survey estimated the worldwide market for transgenic proteins in 2003 to be $2.8
billion (€2.3 billion) growing rapidly to $4.5 billion (€3.7 billion) in 2005 (Jain, 2004). Some of
the products being developed to be produced by GM livestock are conventionally extracted
from human plasma and already have an established market, for example insulin and
fibrinogen.
Table 7 describes in more detail the animals for pharmaceutical production.
Table 7: Description of GM and/or cloned animals for pharmaceutical production
 Description of GM and/or cloned Product                               Estimated Market Size
 animals                                                               (Beltran, 2005)
 GM and cloned Jersey bull has Human               Growth   hormone
 been produced that carries the protein
 human growth hormone gene,
 female offspring of this animals




                                                36
 Description of GM and/or cloned Product                             Estimated Market Size
 animals                                                             (Beltran, 2005)
 will produce hGH in their milk.
 GM      birds   to     produce Alpha interferon
 recombinant Alpha interferon in
 egg white
 GM rabbits                         Recombinant human          C1
                                    esterase inhibitor
 GM and cloned cattle/goats         Recombinant             human
                                    Fibrogen
 GM and cloned cattle/goats         Recombinant             human
                                    lactoferrin
 GM and cloned cattle/goats         Recombinant             human
                                    collagen
 GM and cloned cattle/goats         Recombinant     human anti- $200 million/year
                                    thrombin III
 GM and cloned cattle/goats         Monoclonal antibodies            >$4 billion
 GM and cloned cattle/goats         polyclonal antibodies
 GM and cloned cattle/goats         CD137 antibody
 GM and cloned cattle               Recombinant             human $12 billion
                                    Albumin
 GM and cloned cattle               Recombinant human alpha-1
                                    antitrypsin
 GM and cloned cattle               Recombinant human insulin
 GM goats                           MSP-1 Protein vaccine for
                                    malaria
 GM tilapia                         Human coagulating factor VII     $150-200 million/year
 GM pigs                            Human tissue plasminogen >$1 billion
                                    activator


i. Products already on the market
No products produced in GM animals were identified that are already on the market. Human
antibodies produced in bacteria by phage display are on the market, HUMIRA® an antibody
to TNFα produced as a collaboration between Cambridge Antibody Technology (UK) and
Abbott (US) is approved as a treatment for rheumatoid arthritis in 58 countries including the
EU
ii. Products that may be on the market within 5 years
GM livestock
Between 5-10 products produced in GM animals are progressing through human clinical
trials as part of the regulatory procedures required for pharmaceutical products. Two
products are expected to be on the market in the EU in the next year. This field is expected
to grow substantially over the next 5 to 10 years. Currently 38 out of the top 100 best selling
drugs are proteins and over 700 protein drugs are in development (research director at
company developing pharming products). Furthermore “pharming” may be the only method


                                              37
available to produce the volume to satisfy demand for these protein pharmaceuticals. One
interviewee said 10 approved protein drugs would take up most of the industry’s cell culture
capacity. There was a general feeling that demand for pharmaceuticals produced by GM
animals will increase over the next decade.
Recombinant human anti-thrombin III produced in the milk of transgenic goats is undergoing
Market Authorization Application review with European Medicines Evaluation Agency EMEA.
Recombinant human C1 esterase inhibitor produced in milk of GM rabbits is in Phase III
human clinical trials. Provided the results of the clinical trials and evaluation are positive
these products are likely to be on the market in the next five years.
A number of companies are commercializing production of therapeutic proteins in GM
chicken eggs. Antibodies and therapeutic proteins produced this way are expected to be on
the market in the next 5 years. One of our respondents predicted that over the next 10 – 15
years the patent life of products may be extended by manufacturing them in GM chickens as
opposed to bacterial or mammalian cell culture. It is possible, that manufacturing by avian
transgenics may confer certain advantages (for example increased pharmacological activity)
onto a protein produced using it and therefore create a new IP position for that product, but
this possibility remains untested. Regulatory approval of products produced by this novel
method is seen as the principal hurdle.
Cloned livestock
GM cattle that produce the following recombinant human proteins in their milk at
commercially viable levels have been generated: growth hormone, fibrinogen lactoferrin and
collagen.
Cloning is applied in “pharming” to multiply up GM animals. Using cloning in this way has the
following advantages:
   all animals produced are GM
   all animals produced are all female
   multiple transgenes can be induced
   “mini” herds are created rather than single founders
   development time is reduced
   herd expansion is flexible.
On the assumption that these GM animals will be classified as unfit for human consumption
slaughter animals will be disposed of by incineration. As long as the animals are kept in
strictly controlled containment facilities and are disposed of properly, the question of products
and derivatives should never arise. If derivatives were permitted to enter the human food
chain these will include; semen straws, milk and milk products, beef and beef products,
leather at slaughter, poultry meat, eggs, egg products and other derivatives of eggs, poultry
meat products, and other derivatives of poultry, fish, fish meat, fish meat products, and other
derivatives of fish.
iii. Products that may be on the market beyond 2010
Recombinant human mono- and polyclonal antibodies produced by GM livestock have huge
therapeutic potential for a wide spectrum of clinical indications. GM and cloned cattle have
been produced that express a human chromosome fragment coding for the whole range of
human antibodies. The GM offspring from these GM cloned animals are currently available.
Mono- and polyclonal antibodies produced by GM and cloned livestock are not expected to
be on the market for 5 years. One goal of those commercializing this technology is
production of several different targeted polyclonal antibodies in the same animal. Monoclonal
antibodies from GM cattle for therapeutic use for specific disease indications are not
expected for 10+ years. The priority geographical markets for the antibody products are USA,


                                               38
Japan and Asia and EU. Several technological barriers for this application in cattle were
highlighted: long gestation period in cattle, cloning efficiency and numbers of antibodies
produced.
Two of the significant barriers identified for this application of GM are the time taken to get
the product to market and for the regulatory authorities to “get used to the idea” of
pharmaceutical products from GM animals.
3.4.3 Xenotransplantation
Xenotransplantation is the transplantation of organs or cells from one species to another.
Human to human transplantation has the following problems which could be solved by
xenotransplantation: scarcity of donor organs and need for immunosuppression with adverse
effects of drugs used for this purpose. Pigs are considered the preferred candidate for
xenotransplantation because of physiological compatibility and breeding characteristics.
Large numbers of pathogen free pigs can be raised to provide organs for transplantation into
humans. A major problem with xenotransplantation is hyperacute rejection which appears to
be mediated by regulators of complement activation that are species specific. One approach
to prevent hyperacute rejection is production of GM pigs that express human regulators of
complement activation.
Some of the work in the xenotransplantation field is taking place in public and privately
funded medical facilities. Two countries active in this field of research are USA and Korea.
Xenotransplantation with non GM pig cells is available in Mexico and China (Bioethics New
Zealand , 2005). In 1990’s New Zealand clinical trials were performed implanting non-GM pig
islet cells to produce insulin in diabetes patients, these have been stopped under a
moratorium which has been extended to the end of 2006. The Korean government is
investing large amounts in the xenotransplantation field (60.5 billion won - €0.05 billion) over
the next 10 years with the aim to have a litter of cloned GM pigs to supply organs for human
transplantation by 2010. At least one Korean company has been identified which is
commercialising GM and cloned pigs as organ donors for humans.
There is a chronic shortage of organ donors for patients waiting for transplants. Based on the
identification of a small number of commercial companies developing xenotransplantation of
organs from GM animals this area appears to be more advanced towards commercialisation
than in vitro GM animal cell therapy and tissue engineering, where we were unable to identify
any commercial activity. Currently, approximately 300,000 persons worldwide are awaiting
donor organs. The demand for donor organs has doubled since 1988 and is increasing at a
rate of 15% per year. The value of a transgenic organ is approximately $15,000 to $40,000
(€12,000- 33,000) (Jain, 2004) with a total worldwide market estimated in 2005 at $5 billion
(€4 billion).
Table 8 gives a description of the animals for xenotransplantation.
Table 8: Description of GM and/or cloned animals for xenotransplantation

 Description of     GM      and/or Product                         By-products
 cloned animals
 α-Galactoside GM and cloned Kidneys,    liver,   heart, Cull animals and their
 pigs                        pancreas, organs/cells      derivatives e.g. meat and
                                                         bone meal if rendered, ash
                                                         if incinerated


Aside from the organs and cells produced by the GM and GM and cloned pigs there will also
be derivatives from these animals. On the assumption that these GM animals will be
classified as unfit for human consumption, slaughtered animals will be disposed of by
incineration. As long as the animals are kept in strictly controlled containment facilities and


                                              39
are disposed of properly the products and derivatives should never arise. If derivatives were
permitted to enter the human food chain these derivatives would include meat and bone
meal if rendered, ash if incinerated.
Hyperacute rejection is the first of several immune rejection mechanisms to be overcome
before xenotransplants to humans can be successful. Firstly, an antibody response would be
expected, less vigorous than hyperacute rejection but similar to the response seen in human-
to-human transplants. Second, there will be a cell-mediated response, in which T-cells attack
the xenograft. Finally, xenografts, like human transplants, may be susceptible to chronic
rejection. Chronic rejection is a slow process that happens over months or years and leads to
damage to the blood vessels of the transplant.
i. Products already on the market
We have not identified any examples of xenotransplantation products from GM or cloned
livestock which are currently on the market.
ii. Products that may be on the market beyond 2015
The most optimistic proponents of xenotransplantation think that it will be another 10 years or
so before GM pig organ transplantation will be available, it seems likely that this will first
become available in Asia. The pancreas or pancreatic cells from a GM pig could be the first
to be available commercially followed by kidneys, liver and heart.
3.4.4 Pet sector
Table 9 summarises the application of GM and cloning to the pet sector.
Table 9: Description of GM and/or cloned animals for pets
 Description of GM and/or cloned Product                                On market
 animals
 Fluorescent Protein GM zebrafish          GM pet zebrafish, eggs       Yes
 Cloned cats                               Cloned pet cats              Yes
 Cloned dogs                               Cloned pet dogs              In development
 GM cats with reduced expression of Cats      with     reduced In development
 glycoprotein Fel d 1               allergenicity for humans


i. Products already on the market
The US company Genetics Savings and Clone offer pet cat cloning at $50,000 per animal
and they have cloned 3 pet cats commercially.
GM ornamental fish have been on the market since late 2003 in the United States. The GM
Zebra Danio fish (Brachydanio rerio), with the trade name GloFish™ are sold to ornamental
fish wholesalers by Yorktown Technologies based in Austin, Texas. The fish fluoresce
because they express transgenic red fluorescent protein. Yellow and green varieties are
reported to be in development. The company did not wish to disclose sales volumes or
prices, but the suggested retail price for the fish on the company’s website is $5.00.
They reported current work on sterilization of the fish. This might improve their chances of
marketing the fish outside the US. Both the FDA and the EPA (Environmental Protection
Agency) decided that the fish fell outside their regulatory remit as neither a food nor a threat
to the US environment. The state of California has banned the sale of GloFish.
It is difficult to estimate the potential market size, but Yorktown report that some 200 million
conventional zebra fish have been sold in the US.




                                              40
Fluorescent GM zebrafish are also being sold by a subsidiary of Taikong a aquaculture
business based in Taiwan. Taikong has produced 10 different GM fish, 4 of which are in
mass production and they claim their output is 200,000 fish per month.
ii. Products that may be on the market within 5 years
The physiology of canines makes dog cloning technically more challenging but the dog has
now been cloned by scientists in Korea and Genetics Savings and Clone intend to offer
commercial dog cloning within 5 years.
The US company Allerca is developing hypoallergenic GM pet cats with reduced expression
of glycoprotein Fel d 1. It is claimed that this genetic modification will reduce the allergic
reactions of humans. The price Allerca are quoting for when these animals are available is
$3,500 (€3,000).
3.4.5 Sporting animals
Cloning is being applied commercially on a small scale to rodeo bulls and sport horses.
White-tailed deer have been cloned in Texas because of the commercial value of sport-
shooting of deer.
Table 10 summarises the products available.
Table 10: Description of GM and/or cloned animals for sporting purposes
 Description of GM and/or cloned animals           Product
 Cloned rodeo bulls                                Semen, offspring
 Cloned horses                                     Semen, offspring


Cryozootech is a French company offering cloning of sport horses and endangered equids.
Their priority markets are the EU, Middle East, USA, South America. One of the companies
involved in the commercialisation of cloned livestock has cloned rodeo bulls and is evaluating
the market potential for the cloning of horses.
i. Products already on the market
Two cloned horses have already been sold to private individuals. An unknown number of
cloned rodeo bulls have also been produced for private individuals.
ii. Products that may be on the market within the next 10 and 10+ years
One of our interviewees who is operating in this field expected horse champions to be cloned
for use as stallions within the next 5 years and semen from these to be on the market within
the next 5-10 years. Cloned female champions were also expected to be available in this
longer time frame, followed by offspring of cloned horses in 10+ years.
3.4.6 Endangered species
The main focus of interest in endangered species is the use of cloning to assist reproduction
and save species from extinction where very few individuals remain. A large proportion of the
work on cloning of endangered species has been done either in publicly funded
organizations especially zoos, or as public/private collaborations. Several “frozen zoos”
around the world have biobanked cells from endangered species stored in liquid nitrogen.
i. Products already on the market
A gaur (endangered cattle species) was   successfully cloned by the US company Advanced
Cell Technology (whose current animal    cloning activity is limited to models for biomedical
applications of stem cells). A number    of other species have been successfully cloned
including mouflon, banteng and African   wildcat. Brazil’s Agricultural Research Corporation




                                              41
(EMBRAPA) has successfully cloned the endangered Junqueira cattle and is working on
applying the technology to sheep, pigs and horses.
ii. Products that may be on the market within next 10 and 10+ years
The Chinese Academy of Science’s Institute of Zoology has been working towards cloning
the highly endangered giant panda. The Chinese team has created a panda embryo in an
egg cell from another species and implanted it in the uterus of a surrogate mother, also of a
different species. The final challenge facing them is to achieve development of the embryo in
the womb of another species.
There are a number of limiting factors for cloning of endangered species, principal amongst
them being a source of donor oocytes and the unknown basic reproductive biology to
facilitate reliably oocyte maturation. In some cases the use of cloning by embryo splitting
may be a viable alternative to SCNT cloning for increasing the numbers of endangered
species
In the 1990s, AgResearch in New Zealand cloned the last remaining female of the cattle
‘native’ to Enderby Island. Three cloned heifers were inseminated from the one remaining
bull and with stored semen from dead bulls to produce three heifer calves and resurrect the
breed.
3.4.7 Other possible applications
i. Products that may be on the market beyond 5 years
GM fish are under development for environmental biomonitoring for example for detecting
environmental mutagens. It is unlikely that such technology will be widely used within the
next five years.
CSIRO in Australia are researching the use of GM techniques to produce sterile males of
feral species (for example GM carp) for purposes of population control. In the GM carp an
enzyme which turns the fish female is blocked using antisense technology (New Scientist,
2002). Debate continues as to whether these GM carp should be released into the
environment. However, it is unlikely that such technology will be widely used within the next
five years and may never be part of a commercial business.
A number of participants in our survey indicated that somatic cell nuclear transfer provided a
very good method for facilitating the safe international trade in livestock breeds (or other
species). They considered the technique allowed the transfer of cell lines/tissue and provided
a higher degree of biosecurity than the transport of livestock, embryos or gametes.
One company appears to be commercialising butyrylcholinesterase and an antibody as
countermeasures to bioterrorism. These products may be produced in GM and potentially
cloned animals although it was not possible to confirm this. The research programme to
produce spider silk in GM goats has apparently been discontinued.
3.5 Comparison of economic structure of industry sectors
3.5.1 Food production
The economic structure of the sector is a mixture of SME’s and larger companies with
cloning and or GM expertise. Both SME’s and larger companies are collaborating formally
and informally with large food production companies. With cloning technology there is no
difference in the potential for use by small versus large-scale farmers because the
technology is applied not by the farmer but by the breeding or AI company. GM technology
requires considerable extra paperwork concerning traceability and increased requirements
for containment which make it more difficult for small farmers to apply. GM technology also
raises issues surrounding marketing of products. In the EU small farmers are often members
of co-operatives which may deal with these issues centrally.




                                             42
3.5.2 Molecular pharming
The sector is dominated by SME’s with cloning and/or GM expertise and their collaborations
with larger pharmaceutical or in one case a brewing company. These larger companies have
large R&D portfolios in which the investment in cloning and GM forms a small part. Financial
viability in this sector appears to be a problem, linked undoubtedly to the long time-frame for
getting products to market. It seems that some of the earliest products to market in this
sector will be those that already have an established market, and this will have significant
implications for the sustainability of these businesses.
3.5.3 Xenotransplantation
This sector is dominated by SME’s and start-ups with cloning and/or GM expertise. Some of
these are collaborating both formally and informally with large medical research centres and
hospitals. Financial viability in this sector also appears to be a problem linked undoubtedly to
the long time-frame for getting products to market. Some of the large global pharmaceutical
companies did have some research activity in this sector until recently, but they have
divested this to medical centres. Several of the start-ups in this sector appear to be spin-outs
from universities or research centres.
3.5.4 Pet sector
Apart from the multinational Taikong, the pet sector is made up of SME’s which all have
specialist expertise in cloning and/or GM. At least one of these SME’s is a private company
financed by a single individual.
3.5.5 Sporting animals
Only two companies were identified cloning sport animals. The scale of activity is very small
but high value and apparently unregulated.
3.5.6 Endangered species
As already described most of the activity in this sector is in publicly funded research
organizations especially zoos or in collaborations between these and specialist SME’s.
Government funding is supporting several of the endangered species cloning programmes.




                                              43
SECTION 4 TECHNO-ECONOMIC BARRIERS TO GM AND CLONING
4.1 Main technical barriers
One of the most prominent technical barriers to the use of GM livestock in food production is
that of introgression problems relating to genetic lag. The problem is that the genetic change,
either through addition of a transgene in a GM animal or through cloning of a specific
genotype must then be integrated (introgressed) back into the breeding herd. Selection of the
breeding herd, on a multiplicity of traits, is occurring on a continual basis. Therefore, the most
desirable animal to-day will be overtaken by animals born after it. Thus, the cloned and GM
change must be of sufficient magnitude to better these continuous incremental
improvements.
The time taken to introgress the GM/cloned genotype into the elite population will depend on
transgene effects, initial genetic lag, the risk of inbreeding depression and structure of the
commercial population. It has been predicted that transgene effects of 3-10% of overall
economic merit of the animal will be required to compete genetically with existing genetic
improvement programmes. It is difficult to see what engineered change will generate this.
Thus it is unlikely at the moment that the combination of these technologies will be of
widespread use in an agricultural context.
The high fecundity and general breeding strategy of fish makes introgression of GM into fish
production more feasible. Furthermore, the improvement in growth of GM growth hormone
fish is a step change which surpasses the improvement that could be made through of
conventional selection in one or potentially more generations.
The limitation in fish is that no method for gene targeting is possible. Gene targeting is
possible in livestock through cloning and GM. In birds, there is also no gene targeting
method available. In addition, in birds a method that enables the delivery of large gene
fragments remains to be developed. These limitations are being investigated and current
approaches, e.g. using recombinase systems, are likely to delivery useful tools within 5
years.
The foremost challenges for cloning animals are to increase the efficiency and to overcome
fully Large Offspring Syndrome (LOS). Much progress has been achieved in both, and some
commercial sources question the need for further efficiency improvements. One line of
research is attempting to simplify manipulation methods, e.g. hand made cloning, which in
turn may prelude the automation of the procedure (within 5 years). It is predicted that
improvements in these areas could come from anywhere in the world, with the Far East, USA
and Europe currently leading the way.
Improvements in the actual methodology (Dolly technique) will probably lead to some
substantial advances. Getting transplantable embryos and a higher rate of initiated
pregnancies is now not the main problem. The technique has progressed in terms of yield of
blastocysts from reconstructed embryos. Developmental arrest at the foetal stage remains
the key point questioning the basic issue of the link between robustness (of a complex living
system) and time (reprogramming). A considerable amount of basic research needs to be
carried out before we can expect improvement (within 5 years) and epigenetics is obviously
an important part of this endeavour.
Through the use of nuclear transfer gene targeting, genetic modification in animals has been
accomplished. However, due to the cost involved this method is restricted to high-value
projects by organisations that have substantial financial input. This limits its use in the
academic world. Any technological advance which reduces the cost, which will reflect a
reduction in the number of animals used and hence has positive welfare implications, will
broaden the use of GM and cloning (5-10 years). New methods based on viral vectors can
contribute (within 5 years) but are themselves limited. We can anticipate the use of
innovative approaches, for example based on recombinases, to be the next advance in GM
(within 5 years). One key goal is a “clean” genetic change that allows specific DNA


                                               44
sequences to be altered without the remaining presence of selection marker genes or other
vector sequences. The expectation is that these advances could be generated anywhere in
the world.
Table 11: Key research priorities in cloning and GM
    Cloning and GM research priorities        Significant progress anticipated:
    Overcome LOS                              within 5 years
    Use as basic research tool                within 5 years
    Develop novel     gene      manipulation within 5 years
    strategies
    Derive livestock ES cells                 within 5-10 years


There is however, general agreement within the scientific community that the main barrier to
research is public opinion. Public opinion reflects:
     impact of GM crops on consumer acceptance
     small versus large farming political forces
     political leadership exhibiting suspicion of cloning and GM
     influence of pressure groups, including NGOs and those that have conflicting economic
      interests, e.g. the organic farming community
     uncertainty in the scientific community on the best methods to use
     lack of commercial ventures using cloning or GM that have products for sale, use on the
      farm or within the clinic
     expensive research due to the initial investment needed to set up lab/train people
     risk of escape of cloned/GM organisms . This is likely to be more of a risk for fish than
      terrestrial mammals.
In turn these affect political will and hence public funding support for cloning and GM. Thus
there are only a few groups within a given country that pursue research into cloning and GM.
This then results in little internal competition for funding that in turn results in little funding
allocated to this topic.
4.2 Main barriers to commercialisation
4.2.1 Cloning
Two differing views were expressed regarding the techno-economic barriers to cloning of
food animals. The companies and some researchers believed there were few technical or
economic barriers. The technology works and has been sufficiently improved in efficiency to
be economically viable. One company described the efficiency as now very similar to IVF.
Two research groups said that one of the reasons they had reduced active research on
cloning was because it was now a technology that could be developed for commercial use by
industry.
Conversely, some of the same researchers and others expressed concern that efficiency
was still rather low, that there were significant welfare issues with the process of cloning and
that there was still much to learn about the long-term viability of clones and the impact of
abnormal epigenetic reprogramming. Several research groups around the world are using
cloning of livestock as a means to study epigenetic processes.
In pet cloning the particular reproductive physiology of the dog is a barrier to applying cloning
technology. This is also a barrier for cloning of endangered species where their unknown


                                                    45
reproductive physiology makes it difficult to provide a reliable oocyte maturation environment.
A source of donor oocytes from endangered species is also a considerable barrier to the
application of cloning.
Where clones are to be used as sires of the productive livestock generation, the total
numbers of clones will be relatively small. It will obviously be necessary for these animals to
be fertile and sufficiently fit to have a productive life in an AI stud. If there are problems of
animals with reduced fitness, these will likely be identified early in life. Problems that develop
later in life might be more difficult to detect. There is significant commercial interest within
companies to use cloning in pig production. The reason for the greater interest in cloning
from the pig production than cattle production is because the number of doses of semen from
a boar at AI is limiting whereas this is not the case for a bull. Cloning offers the potential to
increase substantially the number of inseminations possible from a high merit boar by
copying him. Issues of genetic lag are not important for cloned but not genetically modified
animals as there is no need to introgress new genes into an entire population.
There are also risk management issues with the widespread use of clones. Inherited
diseases, for example, could be propagated by the widespread use of a carrier clone and,
consequently good surveillance procedures will be important.
The economic barrier to entry into animal cloning is low. The necessary capital costs are low
and a service company could operate a cloning business with a relatively small, though
highly skilled workforce. It is not yet clear what the costs of obtaining regulatory approval will
be, but it is a reasonable assumption that these costs will be substantially lower than for GM
livestock. The costs for cloning are lower compared with GM because with cloning toxicity
testing is not required, the risk assessments have been undertaken and approval is required
for the process rather than for each individual product. Each new GM product will require
independent approval. In addition, with cloning it takes less time to generate the numbers of
animals required for testing in comparison with conventional breeding of GM animals.
Another factor reducing costs for animal cloning is that there are currently no special
containment regulations for clones so regulatory approval for releases to the environment are
not necessary.
One clear message from the survey undertaken is that the main barrier to the uptake of
cloned sires in the United States is ‘approval’ from the Food and Drug Administration (FDA).
As part of the ongoing process undertaken by the FDA to consider publicly animals cloning
on October 31, 2003, the FDA released a draft executive summary of its assessment of food
and health aspects of animal cloning (FDA News, October 31, 2003). This draft risk
assessment evaluated the effects of cloning on the health and safety of food products from
cloned animals. The report concluded that products from the progeny of healthy clones are
likely to be as safe as those from conventional animals. Final guidance from FDA is now long
awaited. In at least one case, the type of study that might be needed for FDA ‘approval’,
comparing the progeny of clones and control animals, is already underway.
4.2.2 GM
Few of the respondents to our questionnaire highlighted barriers of a technical nature to the
commercialisation of GM animals. The barriers they described were linked to regulatory
approval and consumer/food retailer acceptance.
One of the technological barriers facing commercialisation of GM fish is sterilization
efficiency, currently below 100%. This is important if the fish are to be farmed in sea cages.
The structure and influence of the large retail supermarkets in the EU rather than direct
consumer acceptance was considered a specific barrier to uptake of GM livestock in the EU
by one respondent.
Application of GM technology in the pharming sector has the following technical barriers:
long gestation period in cattle, low cloning efficiency and limited number of antibodies that
can be produced. It is also a technical challenge to produce several different polyclonal


                                               46
antibodies in the same animal. These also constitute barriers for commercialisation. One
respondent indicated that in pharming every new pharmaceutical product produced in GM
livestock has its own unique technical barriers. Regulatory approval of pharmaceutical
products produced by GM animals is seen as the principle hurdle followed by the time to get
the product to market.
Each sector where GM and cloning are being applied presents different ethical and social
challenges. A barrier to attracting venture capital to start-up businesses working in this area
of science is the controversial nature of the work. Furthermore, the IP situation for cloning
technology is complicated and, in an endeavour to simplify this, some of the larger
companies have formed separate holding companies which own and manage the IP.
The relevant Intellectual Property in both the GM and ‘pharming’ sectors is in a relatively
small number of hands. This limits the potential for many new companies to enter the sector
in the medium term.




                                              47
SECTION 5 COMPARISON OF EU WITH NON-EU COMPETITORS
5.1 Current research activities in the EU
Academic research into cloning and GM has followed three themes.
   First the technology itself with two research goals: application to an ever increasing range
    of animal species and testing of technical improvements.
   Secondly, the application of cloning and GM to increase our understanding of the
    molecular events underlying biological process in animals.
   Lastly, to demonstrate what this technology can be used for.
With regard to the last point there has been a raft of proposed uses, largely demonstrated in
the mouse, with some demonstrated in livestock. The majority of these have remained in the
academic area with only a few currently being developed commercially, most notably the use
of cloning to reproduce selected individual animals (endangered, pet or valuable breeding
stock), the generation of animal organ donors for xenotransplantation and the use of animals
as bioreactors.
Currently, the majority of research in cloning and GM within Europe concerns improving the
methodology used in these technologies. In parallel a few commercial ventures have been
established that use this technology.
5.1.1 Summary of EU position
Cloning and GM research within Europe are mainly at a national level. Across Europe it is
fragmented.
Not all member countries have an active research base in cloning and GM of livestock (many
use GM mice for scientific research).
Within a given country the general rule is for one group to have expertise in both cloning and
GM technology.
Within European groups there is considerable cloning and GM expertise and capability;
(currently) at least equal to the rest of the world.
At the moment, Europe has the lead with respect to GM birds.
5.1.2 Summary of international perspective
Research into cloning and GM outside of Europe is largely restricted to the USA and
Australasia.
There is more commercial support for cloning and GM out with Europe than within it. The
greatest contracts is with North America where there are; i) a higher proportion of the
commercial investments in truly commercial companies and, ii) there are commercial farming
companies keen to see the approval of the use of clones as sires of commercial meat
animals.
The USA is moving towards a more centralised approach through initiatives such as the
National Swine Resource Centre (a one-stop site for GM, cloning and embryology in pigs).
Research in the Far East – specifically China, Japan and Korea – is exploding in the area of
cloning and GM. If the increase in effort is maintained, within a few years the majority of
research into cloning and GM will be performed in this region.
The majority of GM fish effort is in the Far East and USA.
5.2 Current commercialisation activities in the EU
Within the EU France is the country which seems to be most actively commercialising GM
and cloned animals. French companies are applying GM and cloning to pharming, sporting



                                              48
and endangered animals sectors. The Netherlands and UK each have one company active in
the pharming sector.
5.3 Non-EU countries closest to commercialising GM and cloned animals
In terms of number of companies and scale of activity, the US is closest to commercialising
the products of GM and cloned animals. Canada is second but with significantly less activity
than the US. From the rest of the non-EU countries listed we could identify only one
company in each commercialising products from GM and cloned animals.
Countries listed in order of total number of cloning/GM companies
   USA
   Canada
   China, Taiwan, Australia, New Zealand, Argentina, Japan, Korea, Cuba, Brazil
We experienced significant difficulty collecting information on the commercialisation activity
in the Far East. The number of companies in USA and Canada reflects the strong
entrepreneurial culture, availability of early stage funding and strength of the public sector
research base.
Non-EU countries are applying GM and cloning to a broad spectrum of possible application
sectors. The most significant difference between EU and non-EU countries is the focus of
non-EU countries on the application of GM and cloning to food production. The impression
given in USA and Canada was of companies poised and ready for the regulatory “green light”
at which point some of these products will be released to the food production market. In
contrast the companies in EU countries are applying GM and cloning principally to pharming
but also to sporting or endangered animals.




                                             49
SECTION 6 REFERENCES
6.1 References
Agrifood News Archive http://www.afaa.com.au/news/news-1380.asp
Bioethics New Zealand, 2005 http://www.bioethics.org.nz/publications/bioethics-progress-
      report-may05/html/page6.html
Beltran, D. (2005) Animal ‘Pharming’ market survey report by Genesis Faraday for DTI, UK
Campbell K.H., Loi P., Otaegui P.J., Wilmut I. (1996) Cell cycle co-ordination in embryo
    cloning by nuclear transfer. Reviews of Reproduction 1,40-46
Campbell K.H., McWhir J., Ritchie W.A., Wilmut I. (1996) Sheep cloned by nuclear transfer
    from a cultured cell line. Nature 380, 64-66
Chan C. 2001. ‘Farmers leap forward with cloned livestock order’, South China Morning Post,
     22 May 2001
Clark A.J. and Whitelaw C.B.A. (2003) A future for transgenic livestock. Nature Review
     Genetics 4, 825-833
Davis, S. (2005) Animal Cloning – Applications and commercial issues. Presentation at the
     workshop Animal Cloning: Technology, Applications and Ethics
European Commission (2004) Report from the Commission to the European Parliament, the
     Council and the European Economic and Social Committee. Life Sciences and
     Biotechnology – A Strategy for Europe. Second Progress Report and Future
     Orientations. 7.4.2004.COM(2004)250 final
Faber, D. C., Ferre, L.B., Metzger, J., Robl, J.M. and Kasinathan, P. (2004) Agro-economic
     impact of cattle cloning. Cloning and Stem Cells. 6 198-207
FDA News, October 31, 2003, U.S. Food and Drug Administration Center for Veterinary
    Medicine. (2003) Animal cloning: A risk assessment. Draft executive summary
    http://www.fda.gov/bbs/topics/NEWS/2003/NEW00968.html
Fischer R., Stoger E., Schillberg S., Christou P. and Twyman R.M. (2004) Plant-based
     production of biopharmaceuticals. Curr Opin Plant Biol. 7: 152-158.
Fricke and Shaver, (2004) Managing Reproductive Disorders in Dairy                  Cows,
      http://www.wisc.edu/dysci/uwex/rep_phys/pubs/MngReproDisorders.pdf
Gordon J.W., Scangos G.A., Plotkin D.J., Barbosa J.A., Ruddle F.H. (1980) Genetic
     transformation of mouse embryos by microinjection of purified DNA. Proceedings of the
     National Academy of Science USA 77, 7380-7384
Hammer R.E., Pursel V.G., Rexroad C.E. Jr, Wall R.J., Bolt D.J., Ebert K.M., Palmiter R.D.,
    Brinster R.L.. (1985) Production of transgenic rabbits, sheep and pigs by
    microinjection. Nature 315, 680-683
Jain, K. K. 2004. Animal biotechnology; technologies, companies and markets. Jain
      Pharmabiotech. Basel, Switzerland
Kaneko T, Moisyadi S, Suganuma R, Hohn B, Yanagimachi R, Pelczar P.
(2005) Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection.
     Theriogenology 64, 1704-15
Kuroiwa Y., Kasinathan P., Matsushita H., Sathiyaselan J., Sullivan E.J., Kakitani M.,
     Tomizuka K., Ishida I., Robl J.M. (2004) Sequential targeting of the genes encoding
     immunoglobulin-mu and prion protein in cattle. Nature Genetics 36, 775-780




                                            50
Loi P., Ptak G., Barboni B., Fulka J. Jr, Cappai P., Clinton M. (2001) Genetic rescue of an
      endangered mammal by cross-species nuclear transfer using post-mortem somatic
      cells. Nature Biotechnology 19, 962-964
Lonergan P., Rizos D., Gutierrez-Adan A., Fair T. and Boland M.P. (2003) Effect of culture
     environment on embryo quality and gene expression - experience from animal studies.
     Reprod. Biomed Online 7, 657-663.
Love J., Gribbin C., Mather C., Sang H.. (1994) Transgenic birds by DNA microinjection.
     Biotechnology (NY) 12, 60-63
Martin, P.A., Crowther, S., Watson, P., Frost, R., Benedictus, J., Enzing, C. (2000)
      Mammalian Cloning in Europe: Prospects and Public policy. Final Project Report.
McCreath K.J., Howcroft J., Campbell K.H., Colman A., Schnieke A.E., Kind A.J. (2000)
    Production of gene-targeted sheep by nuclear transfer from cultured somatic cells.
    Nature 405, 1066-1069
McGrew M.J., Sherman A., Ellard F.M., Lillico S.G., Gilhooley H.J., Kingsman A.J.,
    Mitrophanous K.A., Sang H. (2004) Efficient production of germline transgenic chickens
    using lentiviral vectors. EMBO Reports 5, 728-733
New Scientist, 2002 http://www.newscientist.com/article.ns?id=dn2255
Norman, H. D. et al., (2004) Performance of Holstein Clones in the United States. J. Dairy
    Science 87:729-738
Ristevski S. (2005) Making better transgenic models: conditional, temporal, and spatial
      approaches. Molecular Biotechnology 29, 153-63
Roosevelt, M. 2005. “Would you eat a clone?” Time Magazine, 13 June, 2005.
Schnieke A.E., Kind A.J., Ritchie W.A., Mycock K., Scott A.R., Ritchie M., Wilmut I., Colman
     A., Campbell K.H. (1997) Human factor IX transgenic sheep produced by transfer of
     nuclei from transfected fetal fibroblasts. Science 278, 2130-2133.
Seamark, R. F. March 2003. Review on the current status of the extent and use of cloning in
    animal production in Australia and New Zealand.
Sullivan E.J., Kasinathan S., Kasinathan P., Robl J.M., Collas P. (2004) Cloned calves from
      chromatin remodeled in vitro. Biology of Reproduction 70, 146-153
Swissinfo 24th May 2005 http://www.nzz.ch/2005/05/24/eng/article5812991.html
Tsukui T., Kanegae Y., Saito I., Toyoda Y. (1996) Transgenesis by adenovirus-mediated
     gene transfer into mouse zona-free eggs. Nature Biotechnology 14, 982-985
Vajta G., Kragh P.M., Mtango N.R., Callesen H. (2005) Hand-made cloning approach:
      potentials and limitations. Reproduction, Fertility and Development 17, 97-112
Wells D.N., Misica P.M., Tervit H.R., Vivanco W.H. (1998) Adult somatic cell nuclear transfer
     is used to preserve the last surviving cow of the Enderby Island cattle breed.
     Reproduction, Fertility and Development 110, 369-378
Whitelaw, C.B.A .(2004) Transgenic livestock made easy. Trends in Biotechnology 22, 157-
     159
WHO (2005) Modern Biotechnology, human health and development: an evidence-based
   study
Whittemore, Colin T. (1998) The science and practice of pig production 2nd ed. Oxford :
      Blackwell Science.
Wilmut I., Schnieke A.E., McWhir J., Kind A.J., Campbell K.H. (1997) Viable offspring derived
     from fetal and adult mammalian cells. Nature 385, 810-813.



                                             51
Wrenzycki C. and Niemann H. (2003) Epigenetic reprogramming in early embryonic
    development: effects of in-vitro production and somatic nuclear transfer. Reprod.
    Biomed. Online 7, 649-656.
6.2 Bibliography
Baguisi A., Behboodi E., Melican D.T., Pollock J.S., Destrempes M.M., Cammuso C.,
     Williams J.L., Nims S.D., Porter C.A., Midura P., Palacios M.J., Ayres S.L., Denniston
     R.S., Hayes M.L., Ziomek C.A., Meade H.M., Godke R.A., Gavin W.G., Overstrom
     E.W. and Echelard Y. (1999) Production of goats by somatic cell nuclear transfer.
     Nature Biotechnology 17, 456-461.
Campbell K.H,. Alberio R., Choi I., Fisher P., Kelly R.D., Lee J.H. and Maalouf W.(2005)
    Cloning: eight years after Dolly. Reprod. Domest. Anim. 40, 256-268.
Galli C., Duchi R., Crotti G., Turini P., Ponderato N., Colleoni S., Lagutina I .and Lazzari G.
      (2003) Bovine embryo technologies. Theriogenology 59, 599-616.
Gomez M.C., Jenkins J.A., Giraldo A., Harris R.F., King A., Dresser B.L. and Pope C.E.
    (2003) Nuclear transfer of synchronized african wild cat somatic cells into enucleated
    domestic cat oocytes. Biol. Reprod. 69, 1032-1041.
Heyman Y. (2005) Nuclear transfer: a new tool for reproductive biotechnology in cattle.
    Reprod. Nutr. Dev. 45, 353-361.
Kues W.A. and Niemann H. (2004) Trends Biotechnol. 22, 286- 294.
Lanza R.P., Cibelli J.B., Diaz F., Moraes C.T., Farin P.W., Farin C.E., Hammer C.J., West
     M.D. and Damiani P. (2000) Cloning of an endangered species (Bosgaurus) using
     interspecies nuclear transfer. Cloning 2, 79-90.
Maga E.A. (2005) Genetically engineered livestock: closer than we think? Trends Biotechnol.
    23, 533-535
Lai   L. and Prather R.S. (2002) Progress in producing knockout                    models    of
      xenotransplantation by nuclear transfer. Ann. Med. 34, 501-506
Niemann H., Rath D. and Wrenzycki C. (2003) Advances in biotechnology: new tools in
    future pig production for agriculture and biomedicine. Reprod. Domest Anim. 38, 82-89
Olive V. and Cuzin F. (2005) The spermatogonial stem cell: from basic knowledge to
      transgenic technology. Int. J. Biochem. Cell Biol. 37, 246-250
Piedrahita J.A. and Mir B. (2004) Cloning and transgenesis in mammals: implications for
     xenotransplantation. Am. J. Transplant. 4 Suppl. 6, 43-50
Polejaeva I.A. (2001) Cloning pigs: advances and applications. Reprod. Suppl. 58, 293-300
Prather R.S., Hawley R.J., Carter D.B., Lai L. and Greenstein J.L. (2003)Transgenic swine
     for biomedicine and agriculture. Theriogenology 59, 115-123
Renard J.P., Zhou Q., LeBourhis D., Chavatte-Palmer P., Hue I., Heyman Y. and Vignon X.
     (2002) Nuclear transfer technologies: between successes and doubts. Theriogenology
     57, 203-222
Schatten H., Prather R.S. and Sun Q.Y. (2005) The significance of mitochondria for embryop
     development in cloned farm animals. Mitochondrion 5, 303-321
Sutovsky P. and Prather R.S. (2004) Nuclear remodeling after SNCT: a contractor's
     nightmare. Trends. Biotechnol. 22, 205-208
Thompson AJ, Marques MM and McWhir J (2003) Gene targeting in livestock. Reprod.
    Suppl. 61, 495-508




                                              52
Wang B. and Zhou J. (2003) Specific genetic modifications of domestic animals by gene
    targeting and animal cloning. Reprod. Biol. Endocrinol. 1, 103
Wells D.N. (2005) Animal Cloning: problems and prospectives. Rev. Sci. Tech. 24, 251-264
Wheeler M.B. (2003) Production of transgenic livestock: promises fulfilled. J. Anim. Sci. 81
    Suppl 3, 32-37
Whitelaw C.B.A. and Sang H.M. (2005) Disease-resistant genetically modified animals. Rev.
     Sci .Tech. 24, 275-283
Wilmut I., Beaujean N., de Sousa P.A., Dinnyes A., King T.J., Paterson L.A., Wells D.N. and
     Young L.E. (2002) Somatic cell nuclear transfer. Nature 419, 583-586




                                            53
SECTION 7 GLOSSARY
Adenovirus                                          Chromatin transfer
A group of DNA containing viruses which             A novel system for cloning. In this method
cause respiratory disease in animals,               the chromatin of the donor somatic cell
including one form of the common cold.              nucleus is altered (remodelled) through
These viruses can be genetic modified -             removal of some component of chromatin.
by removing, adding or altering part of
                                                    Differentiation (cell differentiation)
their DNA - and used in gene therapy to
treat disease.                                      This is a concept from developmental
                                                    biology describing the process by which a
Blastocyst
                                                    given    cell    acquires     a    specific
During early mammalian (including                   morphological characteristic (type), e.g.
human)      development     the     zygote          shape, function, composition. The genetic
undergoes cleavage increasing the                   material does not change, rather the
number of cells. When the number                    specific sets of genes that are active
reaches 20-150 cells, a central fluid filled        change. This is associated with changes in
cavity is formed. This stage in the                 chromatin.
development of the embryo is the
                                                    Embryonic stem cells
blastocyst and lasts until the embryo is
implanted into the uterus.                          An embryonic cell that can replicate
                                                    indefinitely (self renewal), transform into
Blastula
                                                    other types of cells (differentiation), and
The generic term for blastocyst in all              serve as a continuous source of new cells.
animals; blastocyst is restricted to                Experimentally defined as being able to
mammalian animals.                                  contribute to all cells of the body during
                                                    the development of an embryo. To date
Breeding pyramid
                                                    the only true embryonic stem cells
The use of a few selected elite animals to          (relating to experimental criteria above)
produce many production animals. The                have been identified in the mouse. Human
genetic and economic merit of the elite             embryonic stem cell lines have been
animals is greater than that of the                 generated and shown to differentiate into
production animals. Current animal                  certain cell types while grown in culture in
breeding tries to maximise the merit of the         the laboratory. Often termed ES cells.
production animals.
                                                    Enucleation
Centrifugation
                                                    The process of removing the nucleus from
The separation of factors through                   an egg. A prerequisite step in cloning by
spinning, usually in a large armoured               nuclear transfer.
machine commonly found in research
                                                    Epigenetics
laboratories.
                                                    The study of chromatin changes to genetic
Chimera
                                                    material, excluding changes to the DNA
A genetic mosaic. An organism in which              itself. Only in rare cases can these
different cells contain different genetic           changes be inherited.
sequences.
                                                    Fibroblasts
Chromatin
                                                    A fibroblast is a cell that can give rise to
The substance of chromosomes that                   other cell types such as bone cells, fat
comprises the DNA and associated                    cells and muscle cells.
proteins in eukaryotic cells. The major
                                                    Gamete
proteins involved are histone proteins.
Changes in chromatin are associated with            The sex cells - egg or sperm.
DNA replication and gene activity.




                                               54
Genome                                              when first introduced into the genome is
                                                    only located on one copy, hence
The whole hereditary information of an
                                                    hemizygous. Such a transgene can be
organism. Mainly composed of DNA
                                                    breed to homozygousity by the mating of,
and/or RNA.
                                                    for example, siblings
Gene targeting
                                                    Heterochromatin
An experimental method based on the
                                                    A type of chromatin that is tightly
process of homologous recombination that
                                                    packaged and usually harbouring inactive
allows specific changes to a target gene
                                                    genes
sequence to be engineered.
                                                    Heterosis
Genetic plasticity
                                                    The increased strength of different
The ability of a cell of one type to change
                                                    characteristics in hybrids, the possibility to
into another type, e.g. fibroblasts can
                                                    obtain a "better" individual by combining
become        fat     cells.    Dramatically
                                                    the virtues of its parents. In plants termed
demonstrated through the birth of 'Dolly'
                                                    hybrid vigour.
who was produced by cloning of a
mammary cell.                                       Homologous recombination
Generation interval                                 The exchange of pieces of DNA that share
                                                    common sequences. Used experimentally
The time between sexual maturity of one
                                                    to introduce a new piece of DNA into a
animal and its offspring.
                                                    genome (the DNA of a cell) and found
Genetic lag                                         naturally during the mixing of genetic
The difference between the genetic merit            material that happens within an egg or
(somewhat equivalent in commercial                  sperm as these cells mature. The latter is
breeding animal to economic merit) of one           termed crossing and allows chromosomes
animal compared to a population of                  to shuffle their genetic material increasing
animals    under    continual  breeding             the potential of genetic diversity.
selection.                                          Imprinted gene
Germ line                                           Imprinted genes are genes whose
Genetic material, DNA and RNA, that                 expression is determined by the parent
comes from the egg or sperm of the parent           that contributed them. In somatic cells, a
that is passed onto the offspring becoming          gene comprises of two alleles; one allele
their germ line.                                    derived from the mother the other from the
                                                    father. Both alleles are usually active,
Gestation                                           however the activity of an imprinted gene
Duration of pregnancy from conception to            is determined by which parent passed on
birth of offspring.                                 which allele. Often associated with
                                                    disease, e.g. Prader-Willi and Angleman
Head-to-tail array                                  syndromes.
DNA constructs (including genes) have a             Integration event
directional property. The arrangement of
two DNA constructs can be in the same or            The process of combining two apiece of
opposite orientation. Head-to tail indicates        DNA into a larger piece of DNA. Used in
that they are in the same orientation.              the context of a transgene recombining
                                                    with the host genome.
Hemizygous
                                                    Introgression
Each gene comprises of two DNA strands,
often termed alleles. If the sequence of            The process of successfully incorporating
each allele is identical this is termed             a specific genotype (combination of allelic
homozygous. If the alleles contain some             types) into a breeding population.
sequence differences (polymorphic) they
are termed heterozygous. A transgene


                                               55
Lentivirus                                           monoploid number. The ploidy of cells can
                                                     vary within an organism. In humans, most
A type of retrovirus, including HIV. These
                                                     cells are diploid (containing one set of
viruses can be genetic modified - by
                                                     chromosomes from each parent), though
removing, adding or altering part of their
                                                     gametes are haploid.
DNA - and used in gene therapy to treat
disease and the generation of transgenic             Primordial germ cells
animals.
                                                     The premature germ cells found in an
Locus                                                embryo.
A genetic region within the genome.                  Pronucleus
Lysostaphin                                          The nucleus of the sperm or egg prior to
                                                     fertilisation.
Lysostaphin is an enzyme (peptidase) that
digests proteins. It specifically destroys           Provirus
certain bacterial cell walls.
                                                     The DNA form of a RNA virus, e.g. a
Methylation                                          retrovirus.
A chemical change where a methyl-group               Quiescent-cell cycle resting stage
is added to a molecule, e.g. DNA, protein.
                                                     Cell growth comprises of a cycle; first
Micromanipulator                                     growth phase (G1), DNA replication (S),
                                                     second growth phase (G2) and cell
A machine used to inject cells, including
                                                     division (M). Given the appropriate
oocytes and zygotes.
                                                     environment or stimuli a cell can exit the
Mosaic                                               cell-cycle and stop growing forming resting
A mixture of something, e.g. gene activity,          phase (G0).
genetic composition (i.e. a chimera).                Reconstructed embryo
mRNA                                                 An embryo that has been formed by
Messenger RNA is a single stranded                   nuclear transfer (cloning).
nuclei acid which is the template for                Recombinant
protein synthesis (that occurs in the
                                                     Through the application of a variety of
ribososme found in the cytoplasm of cells).
                                                     molecular biological techniques new
It is direct copy, produced during
                                                     combinations of DNA and RNA can be
transcription in the nucleus, of parts of one
                                                     generated. Used to study the activity of
strand of DNA.
                                                     genes or produce new proteins.
Nucleus herd
                                                     Recombinase
In many farmed species, genetic change is
                                                     An enzyme that can catalyse the mixing
produced in specific herds and then
                                                     (recombination) of DNA pieces. Usually
disseminated to other farms through
                                                     functions through specific short DNA
transfer of animals, semen or embryos.
                                                     sequences (a consensus sequence). Of
The herds used to produce genetic
                                                     great use in research.
change are known as nucleus herds.
                                                     Re-programming
Oocyte
                                                     The conversion of one cell type into
The female gamete or sex cell.
                                                     another through nuclear transfer (cloning),
Parturition                                          i.e. the conversion of a mammary cell into
The process of giving birth.                         a embryo that resulted in the birth of
                                                     'Dolly'.
Ploidy
                                                     Retrovirus
The number of copies of chromosomes in
a nucleus. The number of basic sets of               A RNA virus that can cause many
chromosomes in an organism is called the             diseases including cancer and AIDS.



                                                56
During infection of a host cell the RNA of           Transgene silencing
the virus is converted into DNA (the
                                                     A phenomenon observed but not clearly
provirus) which then can insert itself into
                                                     understood, although often associated
the cells own DNA (genome). These
                                                     with DNA methylation and other chromatin
viruses can be genetic modified - by
                                                     remodelling events.. Pending on the
removing, adding or altering part of their
                                                     sequences within a transgene and where
DNA - and used in gene therapy to treat
                                                     that transgene is located within the
disease and the generation of transgenic
                                                     genome, the transgene can become
animals.
                                                     inactive or silenced.
RNA interference
                                                     Transposable sequence
A natural process that has been
                                                     A natural class of DNA sequences that
harnessed to experimental use. Specific
                                                     can move from one chromosome site to
small RNA sequences in the correct
                                                     another within the genome. These
configuration can cause the destruction of
                                                     elements can be genetic modified - by
target mRNA sequences thereby affecting
                                                     removing, adding or altering part of their
gene activity patterns within a cell.
                                                     DNA - and used in gene therapy to treat
Somatic cells                                        disease.
All body cells except the reproductive               Transposon
cells, i.e. eggs and sperm.
                                                     A transposable sequence.
Telomerase
                                                     Vector
The enzyme that directs the replication of
                                                     An agent, such as a virus or a small piece
the ends of chromosomes (termed
                                                     of DNA called a plasmid, that carries a
telomeres). These specialised structures
                                                     recombinant or foreign gene. Used for
are involved in the replication and stability
                                                     gene therapy, to generate a transgenic
of chromosomes, associated with the
                                                     animal or deliver DNA to target cells grown
cellular aging process.
                                                     in culture in a laboratory.
Transcriptionally silent
                                                     Zona pellucida
An inactive region of DNA, usually
                                                     The jelly-like structure composed of
associated with a inactive (silent) gene.
                                                     proteins which surround a zygote. It limits
Transcription regulating elements                    access of multiple sperms to the already
                                                     fertilised zygote and prevents viral
Regions of DNA that control the activity of
                                                     infection.
a gene.
                                                     Zygote
Transgenic
                                                     The fusion of sperm and egg results in the
An experimentally produced organism in
                                                     formation of the single cell zygote.
which DNA has been artificially introduced
or changed such that it is incorporated into
the organism's germ line.




                                                57
SECTION 8 APPENDICES
8.1 Appendix 1
Methodology for bibliographic analysis
We found there was no generic database which appeared to give thorough retrieval and
additionally there appear to be inconsistencies in the use of terms. We therefore undertook
searches on several databases and subsequently removed any duplicates. We searched
Articlefirst on Firstsearch and Web of Science. Spot checks indicated that we did not have
thorough coverage of some key journals, so additional searches were carried out through
their respective journal web pages (Nature, Nature Biotechnology, Nature Genetics, Nature
Medicine and EMBO Journal).
Because the different databases provide different search facilities, different search strategies
were adopted.
Articlefirst
Advanced search allows maximum of 3 keywords [with Boolean operators “and” “or” and
“not”
Search terms used were:
   cloning AND animals NOT mouse/mice/rats
   cloned AND animals
   cloning AND cows/cattle/livestock/bovine/calf
   cloning AND cats/kittens
   cloning AND sheep/lamb/ruminants
   cloning AND dogs/puppies
   cloning AND pigs
   cloning AND deer
   cloning AND fish/trout/salmon/tilapia
   cloning AND primates
   cloning AND goats
   cloning AND horses/mules
   cloning AND poultry/chickens
   cloning AND rabbits
   genetic AND modification AND animals
   GM AND animals (The search term ‘GM’ was found to be unhelpful)
   somatic cell nuclear transfer
   transgenic AND animals
Web of Science
Allows truncation, Boolean operators and addition of fields to search. Includes much more
information e.g. authors’ addresses
The search terms used were:
   genetic* AND modif* AND animal* AND journal name [if Nature stable]



                                              58
   clon* AND animal* AND journal name [if Nature stable]
   transgenic AND animal* AND journal name [if Nature stable]
   somatic + cell + nuclear + transfer –mice-mouse-rats
Searches on individual animal name(s) were also used.
The data were manually screened to eliminate:
   Publications relating to ethical issues
   Publications not relevant to the search (e.g. cloned genes)
   Publications which were clearly review articles, although it is likely that some review
    articles remained as they were not clearly identified
Publications were categorised according to species (using the title and abstract where
available) and research area. A very small number of publications referred to more than one
species in which case the paper was categorised according to the first species mentioned.
Where possible, author addresses and affiliations were traced. This was a very time-
consuming exercise and was not possible for all of the publications identified. Publications
were then categorised to a country (based on address of the first named author). Where all
the authors had academic addresses, the publication was categorised as academic. Where
all the authors had an industry address the publication was categorised as industry only.
Otherwise the publication was categorised as joint academic-industry.
With thanks to Moyra Forrest, information officer at Innogen, for her assistance with this
analysis.




                                              59
8.2 Appendix 2
The Cost Benefits of Cloning in a Pig Breeding and Production Systems
The genetic merit of terminal sires used for Artificial Insemination (AI) would be improved
where the terminal sires are clones of high merit animals. Current practice for selection of
terminal sires for AI involves identification of boars with good breeding values from nucleus
herds, but levels of semen production limits the extent of selection and therefore restricts the
genetic merit of boars used. The commercial success of cloning depends on the cost of
cloning AI boars versus the resulting improvements in cost of production in the slaughter
generation.
A simple spreadsheet model of finances in a “typical” European pig breeding and production
operation was set up using the major performance characteristics, cost structures and feed
prices as inputs. Information was obtained from various sources including UK Meat and
Livestock Commission (Pig Cost of Production in Selected EU Countries: Knowles and
Fowler 2004) and trade sources of feed and carcase prices for 2005. This model can be
used to give a general indication of the financial effects of using cloned AI boars. Detailed
predictions for individual countries or production operations would require separate analyses
using appropriate data.
It is assumed that cloning of very high merit boars is to be used instead of the current
practice of selecting boars of good merit. In such a system slaughter generation animals are
not clones, but are related as half sibs (same genetic father, but different mother). Current AI
boars are assumed to represent the top 10% of boars in the nucleus population for both
growth rate and back-fat. Different breeding companies / markets use different economic
indices to identify animals of good genetic merit and it is beyond the scope of this analysis to
make detailed comment on the most appropriate index. However, most breeding
programmes will use indices which place high weighting on growth and back-fat for selection
of sire line pigs.
Sire lines are assumed to be 10% better than the slaughter generation in growth rate (days
to slaughter) and back-fat, and dam lines are assumed to be 10% worse. Coefficient of
variation (CV) for growth and back-fat are assumed to be 12% and 20%, and heritabilities are
assumed to be 0.3 and 0.4 respectively. It is assumed that there will be a genetic lag of one
generation in producing clones to be used at stud. Boars are assumed to be used for 1 year
producing 25 tubes of semen per week which are used at a rate of 2.2 tubes per sow.
The effect of moving from the current practice to using cloned boars is calculated as the
difference between performance of the top 10% and the top 1% or 0.5% for each trait of
boars from the nucleus herd. Whether cloning in practice can achieve selection within the top
1%, 0.5% or even higher depends on the effectiveness of the cloning procedure, the size of
the nucleus herd and the demand from customers – all of which have yet to be determined.
The figures of 1% and 0.5% are thought to be reasonable estimates of what is likely to be
achievable. It is assumed that all animals can be cloned and that there is no effect of the
procedure on the fertility of the cloned boars. If either of these factors were to be significant,
then the cost effectiveness of using cloned boars would be reduced.
The financial model is shown in Table 1. There are likely to be major improvements in cost of
production (Table 2) as a result of using cloned boars of at least €1.18 to €1.68 per pig
produced for slaughter from the top 1% and top 0.5% of nucleus herd boars respectively.
This translates to 1.1% - 2.4% improvement in costs depending on the production system.
The cost benefit to the operation per cloned boar is in the range €6900 - €19000 depending
upon the production system (Table 2). Application of the technology would be financially
worthwhile where the cost of cloning boars is significantly less than this. Assuming that the
European slaughter pig herd totals 243 million (FAS 2005) and that 50% of these pigs were
to be produced using AI then using cloned AI boars would represent a cost of production
improvement of between €143M and €204M for European pig producers.



                                               60
This analysis is based on conservative estimates of improved growth and back-fat levels
achieved by using clones of the fastest growing and leanest boars. The difference between
current average AI boars and clones of top boars may be even greater than used in these
calculations. Furthermore these estimates do not take account of advantages that would be
achieved in the processing chain, from improved saleable yield per carcase and reduced
wastage. Even further financial benefit is likely to be seen by using indices including
additional production parameters appropriate to individual production systems. Identifying
boars with desirable characteristics in meat quality or skeletal integrity would also improve
the cost effectiveness of cloning. An approach such as this would be very efficient in
producing boars for “niche” products for premium quality markets. Equally, such an approach
could also be adapted to make maximum use of exceptionally high performing animals in
demand by a number of customers / markets.
Use of cloned AI boars would reduce the number of boars being used in commercial
production operations from typical levels of 10 to 50 per operation to effectively less than 5,
or even 1. It is likely, however that different operations and different countries / markets
would require different boars for their own particular needs. This procedure for use of cloned
boars is analogous to the traditional practice of identifying the best boar for use on an
individual farm and using only this boar for a period of time. Slaughter generation animals are
half sibs with variation in the slaughter generation being maintained within the dam lines.
Breeding companies would still have to maintain nucleus herds of the current size to ensure
adequate control over inbreeding. This technology would not be appropriate to a nucleus
herd since the use and rapid turnover of a number of high merit boars is required to generate
the genetic variation required to produce subsequent generations.
A number of economic factors are likely to affect pork production in the next decade. Factors
which reduce the cost of production such as CAP reform reducing the cost of feed act to
reduce the cost advantages of using cloned boars in absolute but not relative terms. Factors
which increase the cost of production such as increased cost of disposing of waste will
increase the cost advantages of using cloned boars in absolute but not relative terms.




                                              61
Table 1. Performance and cost in a typical European pig production operation. Performance
is predicted for a base population using AI (from top 10% boars) and for breeding herds
using AI from cloned boars deriving from the top 1% and top 0.5% of the nucleus population
for days to slaughter and back fat depth.


 Parent Generation                        Base      Top 1%     Top 0.5%
 Number of litters per year               2.23      2.23       2.23
 Pigs born alive per litter               11.16     11.16      11.16
 Piglet mortality %                       11.65     11.65      11.65
 Pigs reared per litter                   9.86      9.86       9.86
 Pigs reared per sow per year             21.99     21.99      21.99
 Sow feed consumption tonnes              1.195     1.195      1.195
 Sow feed cost €/tonne                    157.68    157.68     157.68
 Feed cost per sow €                      188.43    188.43     188.43
 feed cost per piglet €                   8.57      8.57       8.57
 Slaughter Generation                     Base      Top 1%     Top 0.5%
 Age days                                 166.0     164.4      163.7
 Live weight kg                           111.00    111.00     111.00
 Backfat mm                               12.00     11.65      11.54
 Killing Out % (from Whittemore 1998)     77.43     77.39      77.37
 Dead weight kg                           85.947    85.901     85.885
 Lean Meat% (from Whittemore 1998)        58.23     58.63      58.76
 Lean Meat kg                             64.638    65.082     65.226
 FCR                                      2.830     2.790      2.773
 Feed Consumption / day @ 100kg           2.8
 Feed Consumed (kg)                       314       310        308
 Grower feed €/kg                         178.38    178.38     178.38
 Costs                                    Base      Top 1%     Top 0.5%
 Sow feed cost per piglet €               8.57      8.57       8.57
 Growing feed cost €                      56.03     55.24      54.90
 Other Variable costs €                   14.01     13.87      13.81
 Other Variable costs / day €             0.08
 Fixed Costs €                            25.67     25.42      25.32
 Fixed Costs / day €                      0.15
 Total Cost per slaughter pig €           104.28    103.10     102.60
 Improvement per slaughter pig €                    1.18       1.68




                                           62
Table 2. Costs of production in a typical European operation. Costs are calculated for a base
population using AI and for breeding herds using AI from cloned boars deriving from the top
1% and top 0.5% of the nucleus population for days to slaughter and back fat depth.


 Cost of Production                           Base     Top 1%      Top 0.5%
 Cost of production €/kg lwt                  0.939    0.929       0.924
 Cost of production €/kg dwt                  1.213    1.200       1.195
 Cost of production €/kg lean meat            2.084    2.047       2.033
 Improvements
 Cost of production €/kg lwt                           0.011       0.015
 Cost of production €/kg dwt                           0.013       0.019
 Cost of production €/kg lean meat                     0.037       0.051
 Improvements %
 Cost of production €/kg lwt %                         1.13        1.61
 Cost of production €/kg dwt %                         1.08        1.54
 Cost of production €/kg lean meat %                   1.75        2.43
 Improvement per Stud Boar                    Base     Top 1%      Top 0.5%
 Cost of production per pig (live)            104.28   103.10      102.60
 Improved cost per pig (live) €                        1.18        1.68
 Improved cost € per boar @ 5826
 pigs/boar/y                                           6878.07     9796.43
 Improvement per Stud Boar                    Base     Top 1%      Top 0.5%
 Lean meat kg / carcase                       64.638   65.082      65.226
 Improved cost of production of lean meat /
 pig €                                                 2.38        3.30
 Improved cost € per boar @ 5826
 pigs/boar/y                                           13852.00 19234.13




                                              63
8.3 Appendix 3




Questions to companies working on animal cloning and/or GM animals
INSTRUCTIONS – completing the form electronically
Please “tick” relevant boxes where the following symbol is shown        by clicking on boxes
using a mouse
Please provide information in boxes         by clicking on the box and typing
INSTRUCTIONS – completing the form by hand
Please tick relevant boxes and write information in boxes. Where insufficient space, please
use extra sheets where necessary, and write the question number your answer corresponds
to.
INFORMATION ON RESPONDANT
 Respondent Name
 Position within the Organisation
BUSINESS DETAILS
 1.A. Please describe your organizations Cloning & GM activities:
 Only animal cloning
 Only GM animals
 Animal cloning AND GM animals
 None of the above
 If None of the above, please state your
 organizations business
 1.B. From these animals, is your organization selling;
 Offspring
 Derived products
 Other (please state)
 2. Which species do you work with?
 Cattle
 Sheep
 Pigs
 Salmon
 Chickens



                                              64
Tilapia
Cats
Goats
Zebrafish
Other (please state)
3. Which of the following target sectors do you work in (if more than 1 sector, please rank in
order of priority, 1 being most important)?
                                             Tick box       Priority number
Food production
Molecular pharming – (producing animals
that   will  produce     pharmaceutical
products)
Xenotransplantation
Pet sector
Sporting animals
Endangered species
Other? please state
4.Does your business currently or intend to offer a business or a service, or both
A service
A product
Both a service and a product
5. What sort of organisation are your actual or expected customers?
Individual pedigree animal breeders
Individual commercial animal breeders
Animal breeding companies
Healthcare providers
Private individuals
Zoos
Other (please state)
6. Which geographical markets are your
priorities?
7. In which countries do you have
products in the market?
8. What is your business model or
structure, how do/will you make your
money?
9. What products do you have on the
market?
And of these products what total value or volume(s) have been sold?



                                             65
<$50,000
$50-200,000
$200,000 – $1m
>$1m
Volume – Please state
10. What is your price model per unit
$1-10
$10-50
$50-200
$200-1000
$1000-5000
$5-10,000
$10-50,000
>$50,000
11. Do you have a price list we could Yes        No
have?
12. What share of your business cash-flow comes from sales?
<5%
5-10%
10-30%
30-50%
>50%
13. How many cloned/GM, cloned and GM animals do you have?
<5
5-10
10-30
30-50
>50
14. What is the cost of producing clones, GM animals or cloned/GM animal?
                    Cloned                GM                   Cloned GM
$1-10
$10-50
$50-200
$200-1000
$1000-5000
$5-10,000




                                          66
$10-50,000
>$50,000
15. How do you see costs evolving in the future;
                               Decreasing          Staying the same    Increasing
Next 5 years (2005-2010)
5-10 years (2010-2015)
10+ years (after 2015)
16. How do you picture the future for your
business?
17.Optional question for food production organizations - When do you think cloned/GM animals
will be affordable for an average farmer?
Next 5 years (2005-2010)
5-10 years (2010-2015)
10+ years (after 2015)
18. What products do you expect to have on the market in the next 5 years (2005-2010) and 5-
10 years (2010-2015), 10+ years (after 2015)?
Product - We would like a next   5    years 5-10 years (2010- 10+ years (after
detailed description of   (2005-2010)       2015)             2015)
(i) animal (ii) primary product
and or ultimate product, i.e. (i)
Cattle (ii) Genetically modified to
produce human growth hormone
19. How do you see your cloning/gm business share evolving in the next 5 years (2005-2010)
and 5-10 years (2010-2015), 10+ years (after 2015)?
                 next 5 years (2005-2010)   5-10 years (2010-2015)     10+ years (after 2015)
Decreasing
Increasing
Other        –
Please state
20. Thinking more broadly about this
sector what products do you see coming
in these timescales?
21. Is there company literature (Brochure/company report) we can have a copy of?
Brochure/company report?
Yes                                         No
22. Do you have publicly available information on the benefits of your products that you could
send us?
Yes                                         No
23. Who are the owners of the company
and what are their expectations?
24. Who are your business/academic



                                            67
collaborators?
25. Do you have other non-cloning non-GM services/products in your portfolio?
No
Yes      If Yes please describe
26. We would like to know what share of your R&D budget%, sales and profit%, total no. of
employees is engaged in animal cloning, gm or both.
GM      and      cloned R&D            sales and Total no. of
animals, or offspring or budget%       profit%   employees
derived products
<5%
5-10%
10-30%
30-50%
>50%
27. We would like to know what share of your business in animal cloning, gm or both is on the
market or in the pipeline?
GM      and      cloned On market      In pipeline
animals, or offspring or
derived products
<5%
5-10%
10-30%
30-50%
>50%
28. What size is your organization?
Turnover
<$50,000
$50-200,000
$200,000 – $1 m
>$1m
No. of staff
<5
5-10
10-30
30-50
50-100
100-200
>200



                                           68
29. How much do you invest in R&D, TT and innovation related to cloning/GM?
<$50,000
$50-200,000
$200,000 – 1 m
>$1m
30. How share of investments in R&D, TT and innovation related to cloning/GM compare with
your other activities?
<5%
5-10%
10-30%
30-50%
>50%
TECHNICAL
31. What do you feel are your
technological barriers remaining to be
overcome?
SOCIAL, REGULATORY AND ECONOMIC IMPACT
32. To what extent do you agree/disagree with the following statement relating to regulatory
issues relating to your product(s)
                                         Agree        Agree       Neither   Disagree     Disagree
                                         strongly                 agree nor              strongly
                                                                  disagree
The regulation in this area is well
defined
There is no regulation in this area
The regulation is confusing
The regulation is excessive
The regulation is inadequate
33. What do you feel are the public attitudes to your product(s)
                              Very         Positive    Neither     Negative   Very       Don’t
                              positive                 positive               negative   know
                                                       nor
                                                       negative
In North America
In Europe
In Asia
In Australia/New Zealand
GENERAL
34. Who else do you think we should talk
to /contact in this area?



                                                69
8.4 Appendix 4
Letter to companies accompanying questionnaire




Dear


Transgenic or cloned animal commercialisation


The European Commission has commissioned a study which aims to anticipate which GM
and/or cloned animals and derived products will request authorisation for commercialisation
in the EU over the next decade. This study is being carried out by the Genesis-Faraday
Partnership, Edinburgh (UK) through the European Science and Technology Observatory
(ESTO) network, in co-operation with the Institute for Prospective Technological Studies
(IPTS), Seville (Spain) from the European Commission Joint Research Centre.
We believe it is important to include your input in this study, I realise that that you are a very
busy person, but assure you that this should not occupy more than 0.5-1 hour of your time.
This research would take the form of a confidential questionnaire, which I include. The
questionnaire will be treated as strictly confidential and only aggregated results will be
presented. We would be grateful if you could please return the questionnaire by June 10th.
Your kind co-operation would be greatly appreciated by both the European Commission and
myself at the Genesis Faraday Partnership.
Yours sincerely




Cecilia Oram
Technology Translator



Genesis Faraday
Roslin BioCentre
Roslin
Midlothian
Scotland
UK
EH25 9PS

tel +44 131 527 4332
fax +44 131 527 4335

cecilia.oram@genesis-faraday.org




                                               70

								
To top