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					S1:44                                     BVAAWF=FRAME=RSPCA=UFAW Joint Working Group on Re nement


be paid to these and approval secured from          in mind, the Sixth BVAAWF/FRAME/
the relevant authorities (e.g. in the UK these      RSPCA/UFAW Joint Working Group on
are the Department for the Environment,             Re nement set out to identify and
Food and Rural Affairs, and the Home Of ce).        document the areas of concern and how
   Con rmation should be given by those             they might be addressed. In doing so, the
sending the mice that they have been                Working Group has identi ed current best
despatched and those receiving the mice             practices that should ensure the number of
should con rm receipt. All GM mice, or fresh        mice used is kept to a minimum and their
or cryopreserved embryos, should be                 welfare improved, without compromising
accompanied by information detailing the            scienti c objectives. The main recom-
microbiological status, the nature of the           mendations are summarized below.
phenotype and any specialist management the
mice require in terms of housing, husbandry
and veterinary care. These arrangements             25.1 Re nement
should be discussed prior to the transport of       Proper design of constructs and choice of
the mice so that appropriate measures are in        animals can re ne the production of GM
place. Prospective discussions should include       mice. The use of inducible promoters and
all of those involved in using and caring for       conditional transgenes can minimize the
the mice.                                           effects of the genetic modi cation on
                                                    animal welfare (see Section 3). The use of
Recommendations:                                    SPF mice prevents incidental infection
° The transport of live mice should be              with pathogenic microorganisms and thus
  replaced, wherever possible, by the use of        avoids the possibility of clinical disease
  fresh embryos, or cryopreserved embryos           and death (see Section 8).
  and gametes.                                         Breeding and husbandry considerations
° Live mice should only be transported by           can re ne methods in the production,
  approved animal couriers and in                   maintenance and transport of GM mice.
  accordance with LABA/LASA guidelines.             Optimizing the size of prepubescent egg
° The nature of the phenotype and any               donors and the careful selection of stud
  specialist care required should be dis-           males can help prevent the females being
  cussed prior to the transport of the mice,        harmed during mating (see Sections 11.1.1
  and detailed in written information               and 12). Pseudopregnant females receiving
  accompanying the GM mice.                         embryos from the same microinjection
                                                    experiment can be housed together after
                                                    embryo transfer, thus avoiding having to
25 Summary
                                                    keep social animals singly (see Section 13.4).
Despite the extensive use of GM mice,               In this case, the females will help each
relatively little has been published regarding      other raise the joint litter. The distribution
applying the principles of reduction and            of cryopreserved embryos, sperm or ova,
re nement to their generation, management,          rather than live mice, avoids the potential
and care. This re ects in part the nature of        welfare problems associated with transport
the technology—the large numbers of mice            (see Section 24).
used both in the generation and breeding of            Re nements in surgical techniques are
GM mice that are of scienti c ‘interest’, the       possible. Vasectomies performed by a
surgery and other potentially painful proce-        scrotal sac incision rather than laparotomy
dures that are involved, and the dif culties        are a re nement as they avoid cutting the
associated with predicting the effects of           abdominal body wall musculature and are,
genetic modi cation. While these factors            therefore, likely to be less painful (see
represent signi cant hurdles, progress has          Section 14.1). The use of genetically sterile
also been limited by a failure to consider,         males as an alternative removes the need to
implement and disseminate best practices            subject males to surgical vasectomy (see
wherever possible. With such considerations         Section 14.2). This re nement, however,
Laboratory Animals (2003) 37 (Suppl. 1)
Re nement and reduction of GM mice                                                                   S1:45


con icts with the principle of reduction            and to ensure that husbandry and care are
since a separate colony of mice has to be           optimal (see Section 17).
maintained. The surgery required for
vasectomy and embryo transfer can cause
pain. Recognizing subtle indicators of pain         25.2 Reduction
can be dif cult in mice and it is                   Unnecessary production and use of GM
recommended that a precautionary                    mice should be avoided. This requires a thor-
approach is adopted for the management              ough search of subject-speci c and specialized
of pain, with pre-emptive analgesia being           databases such as the Trangenic/Targeted
given to all mice undergoing invasive               Mutation database (TBASE, http://tbase.jax.
procedures (see Section 10.4).                      org/), and cryopreservation banks, to ensure
   The removal of biopsies for genotype             that the GM mice are not already available.
analysis can cause pain, suffering and              Comprehensive searches are required to
distress. It is important that the least            determine whether the transgene is suitable
invasive method is used and the size of             in terms of its promoter speci city and
the biopsy taken is kept to an absolute             potential levels of expression (see Section 4).
minimum (see Section 15). The screening                The design of the transgene, in addition to
system used to distinguish GM from                  the strains used for generating a new GM
non-GM mice should seek to minimize the             line, requires careful consideration in order
amount of tissue required. Southern blot            to minimize the production of unwanted
hybridization requires more DNA and                 mice. Inclusion of insulator or intronic
concomitantly more tissue than analysis by          sequences in the transgene can help avoid
PCR, and therefore the use of PCR for               the effects of random transgene integration,
genotyping should always be considered. Tail        thus increasing the likelihood of producing
biopsies are commonly used as a source of           scienti cally ‘informative’ GM mice (see
tissue for genotyping. The removal of even a        Section 4.2). Selection of an appropriate
small section of tail is likely to be painful and   strain as a source of eggs for microinjection
the use of more humane sources of biopsy            can reduce the overall number of mice used
materials, for example, ear tissue, and oral,       to generate a line (see Section 11.1.2). The
faecal or blood samples, should be                  selection of males with high and consistent
investigated as alternatives, particularly          plugging rates should minimize the numbers
where the intention is to use PCR (see              of mice required to generate pseudopregnant
Sections 15.1, 15.3 and 15.4). Where the use        females (see Section 14). Careful choice of
of tail biopsies is unavoidable, no more            the strain of host blastocysts increases the
than 5 mm of tail should be taken and               likelihood of obtaining germline transmis-
appropriate anaesthesia and analgesia should        sion of the ES cell genome (see Section 5).
be used. Unless there is scienti c justi ca-           The number of animals used to produce GM
tion to the contrary, tail biopsies should not      mice can be minimized by ensuring that all
be taken from mice signi cantly younger             staff are given appropriate training and have
than 3 weeks of age or older than 4 weeks of        the specialist skills and knowledge required
age (see Sections 15.2.1 and 15.2.2).               (see Section 6). Monitoring the ef ciency of
   Mice should be marked using non-                 transgenic production by using benchmarks
invasive methods, where possible. Readily             gures as an indicator of performance should
observable variations such as sex and coat          identify where remedial action is necessary
colour should be included in order to               (see Section 6.3). A good understanding of
minimize the number of mice that require            laboratory animal science and husbandry
marking (see Section 16).                           practices are essential for the careful
   Genetic modi cation can have a                   management of colonies, to match supply
deleterious effect on animal welfare. All GM        to demand, and to avoid the production of
mice should be carefully monitored so that          surplus mice.
appropriate action can be taken to minimize            Cryopreservation of gametes, embryos
any harm, to develop humane endpoints               and ovarian tissue provides the opportunity
                                                                    Laboratory Animals (2003) 37 (Suppl. 1)
S1:46                                        BVAAWF=FRAME=RSPCA=UFAW Joint Working Group on Re nement


to archive transgenic lines until required,              Brinster RL, Allen JM, Behringer RR, Gelinas RE,
and thus avoids the potential wastage                       Palmiter RD (1988) Introns increase
                                                            transcriptional ef ciency in transgenic mice.
associated with their maintenance by
                                                            Proceedings of the National Academy of Science
continuous breeding (see Section 19). The                   USA 85, 836–40
cryopreservation of sperm is itself a                    Broome RL, Feng L, Zhou Q, Smith A, Hahn N,
reduction initiative as it substantially                    Matsui SM, Omary MB (1999) Non-invasive
reduces the numbers of mice required to                     transgenic mice genotyping using stool analysis.
store and regenerate a transgenic line                      FEBS Letters 462, 159–60
compared with the cryopreservation of                    Carter RJ, Hunt MJ, Morton AJ (2000) Environmental
                                                            stimulation increases survival in mice transgenic
embryos (see Section 19.2).                                 for exon 1 of the Huntington’s disease gene.
  Provided that no adverse welfare problems                 Movement Disorders 15, 925–37
have been identi ed, homozygotes should be               Charreau B, Tesson L, Soulillou J-P, Pourcel C,
bred (see Section 18). Such breeding                        Anegon I (1996) Transgenesis in rats: technical
programmes prevent the production of                        aspects and model. Transgenic Research 5,
unwanted genotypes, as well as negate the need              223–34
                                                         Costa P (1997) Neuro-behavioural tests in welfare
to genotype and identify mice (a re nement).
                                                            assessments of transgenic animals. In:
Care should be taken, however, as the random                Proceedings of the 6th FELASA Symposium on
integration of transgenes can cause mutations               International Harmonization of Laboratory
with no phenotypic effects in heterozygotes but             Animal Husbandry Requirements. London:
which lead to poor welfare in homozygotes.                  Royal Society of Medicine Press, pp 51–3
                                                         Dennis M (2000) Humane endpoints for genetically
                                                            engineered animal models. Institute for
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