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INTRODUCTION
Gene transfer is to transfer a gene from one DNA molecule to another DNA molecule. Gene
transfer represents a relatively new possibility for the treatment of rare genetic disorders and
common multifactorial diseases by changing the expression of a person's genes (Arat, 2001). In
1928, Griffith reported that a nonpathogenic pneumoccocus strain could become pathogenic
when it was mixed with cells of heat-killed pathogenic pneumoccocus, which hinted that the
pathogenic genetic material could be transformed from the heat-killed pathogenic pneumoccocus
to the nonpathogenic strain (Griffith, 1928). This is the first report for gene transfer observation.
However, the transforming substance was not identified in these experiments. Up to 1944, Avery
et al demonstrated that deoxyribonucleic acid (DNA) was the transforming substance (Avery,
1944). In 1952, Hershey and Chase showed that DNA was the only material transferred during
bacteriophage infection, which suggested that the DNA is the genetic material (Hershey, 1952).
The basic technique for introducing DNA into E. coli have inspired procedures for the
introduction of DNA into cells from a wide variety of organisms, including mammalian cells.
Genetic engineering of food is the science which involves deliberate modification of the genetic
material of plants or animals. Introduction of DNA into plants is of great agricultural potential
and medical importance (Campbell, 1999; Uzogara, 2000; Lorence, 2004).
The gene transfer methods normally include three categories:
1. transfection by biochemical methods;
2. transfection by physical methods;
3. virus-mediately transduction.
The gene transfer results can be transient and stable transfection.
Gene therapy can be defined as the deliberate transfer of DNA for therapeutic purposes. Many
serious diseases such as the tragic mental and physical handicaps caused by some genetic
metabolic disorders may be healed by gene transfer protocol. Gene transfer is one of the key
factors in gene therapy (Matsui, 2003), and it is one of the key purposes of the clone (Ma, 2004).
Gene transfer can be targeted to somatic (body) or germ (egg and sperm) cells. In somatic gene
transfer the recipient's genome is changed, but the change will not be passed on to the next
generation. In germline gene transfer, the parents' egg and sperm cells are changed with the goal
of passing on the changes to their offspring. Germline gene transfer is not being actively
investigated, at least in larger animals and humans (Bordignon, 2003; Umemoto, 2005).
Generally, there are 9 ways for gene transfer:
(1) Lipid-mediated method;
(2) Calcium-phosphate mediated;
(3) DEAE-dextran-mediated;
(4) Electroporation;
(5) Biolistics;
(6) Viral vectors;
(7) Polybrene;
(8) Laser transfection;
(9) Gene transfection enhanced by elevated temperature (Sambrook, 2001).
Agrobacterium Mediated Transformation
Agrobacterium tumefaciens and Agrobacterium rhizogenes are pathogenic soil bacteria that
contain a Ti (tumor inducing) or Ri (root inducing) plasmid. A small piece of this plasmid, the
T-DNA is transferred from the bacteria to the plant and integrates stably into the plant genome.
The focus will primarily be on A. tumefaciens and the Ti-plasmid because it is more common.
The T-DNA encodes genes that stimulate cell division (result in production of plant hormones)
and the synthesis of opines (amino acid derivatives; either nopalines or octopines). These opines
are used by the bacteria as carbon and nitrogen source. The Ti-plasmid contains genes that help
in the break-down of these opines.
The T-DNA (transfer-DNA) is transferred to the host. The T-DNA is flanked by a left and a right
border, consisting of 25 bp direct repeats. These borders are recognized by endonucleases
encoded by the virD1 and virD2 genes, which are part of the Ti-plasmid. The virD2 protein nicks
the border sequence and binds to the 5’ end of the nicked DNA. The T-DNA is thought to
unwind from the plasmid and is then transferred to the plant as a single-stranded DNA molecule.
It is coated with the virE2 protein, which is a DNA binding protein with a nuclear import signal
that targets the T-DNA to the plant’s nucleus, where it integrates into the genome.
The use of this naturally occurring gene transfer process as a transformation tool was made
possible by the discovery that the Ti-plasmid could be disarmed, i.e. the tumor inducing genes
on the T-DNA could be eliminated without affecting the gene transfer itself. So you could
replace the undesirable genes with YFG!
The modern expression vectors can replicate in both E. coli and A. tumefaciens. So we can
construct the plasmid in E. coli, harvest large quantities of it, make sure it is OK, and then
transfer it to A. tumefaciens via electroporation (an improvement over the tri-parental matings
that were used in the beginning).
The actual transformation process involves infecting the plant tissue with A. tumefaciens
carrying the newly designed Ti-plasmid.
The general method involves the use of a leaf punch that is incubated in a suspension of bacteria.
The cells on the edge of the punch are transformed. These transformed cells now need to
develop into new plants through tissue culture. The leaf disk is typically placed on a medium
that stimulates shoots (high cytokinin:auxin ratio), selects for transformants (based on the
selectable marker) and kills Agrobacterium (carbenicillin). The shoots that form are placed on a
root-inducing medium (low cytokinin:auxin ratio) and the resulting plantlets are eventually
transferred to soil.
This regeneration process is very similar for tissues transformed with the particle gun.
The latest Agrobacterium-based transformation technique in Arabidopsis is referred to as “floral
dip”. In this case the tissue culture stage has been eliminated. Instead, the Arabidopsis plant is
dipped into a suspension of Agrobacterium at the stage where it has the maximum number of
unopened floral bud clusters. The suspension also contains a detergent to reduce surface tension,
and some sugar. The bacteria get into the developing flowers and transfer the T -DNA. This then
results in the formation of transgenic seeds that can be planted out on medium with a selectable
marker. The plants that survive on the selectable medium get transplanted and can be studies or
screened.
Some approved transgenic plants are Soybean, Corn, Cotton, Oil Seed rape, Sugarbeet,
Squash, Tomato,Tobacco, Carnations, Potato, Flax, Papaya, Chicory, Rice, Melon.
REVIEW OF LITERATURE
Agrobacterium tumefaciens: a natural tool for plant transformation
Plant transformation mediated by Agrobacterium tumefaciens, a soil plant pathogenic bacterium,
has become the most used method for the introduction of foreign genes into plant cells and the
subsequent regeneration of transgenic plants. A. tumefaciens naturally infects the wound sites in
dicotyledonous plant causing the formation of the crown gall tumors. The first evidences
indicating this bacterium as the causative agent of the crown gall goes back to more than ninety
years (Smith and Townsend, 1907). Since that moment, for different reasons a large number of
researches have focused on the study of this neoplastic disease and its causative pathogen.
During the first and extensive period, scientific effort was devoted to disclose the mechanisms of
crown gall tumor induction hoping to understand the mechanisms of oncogenesis in general, and
to eventually apply this knowledge to develop drug treatments for cancer disease in animals and
humans. When this hypothesis was discarded, the interest on crown gall disease largely
decreased until it was evident that this tumor formation may be a result of the gene transfer from
A. tumefaciens to infected plant cells.
Fig: Disease cycle caused by A.tumefaciens
A. tumefaciens has the exceptional ability to transfer a particular DNA segment (T-DNA) of the
tumor-inducing (Ti) plasmid into the nucleus of infected cells where it is then stably integrated
into the host genome and transcribed, causing the crown gall disease (Nester et al., 1984; Binns
and Thomashaw, 1988). T-DNA contains two types of genes: the oncogenic genes, encoding for
enzymes involved in the synthesis of auxins and cytokinins and responsible for tumor formation;
and the genes encoding for the synthesis of opines. These compounds, produced by condensation
between amino acids and sugars, are synthesized and excreted by the crown gall cells and
consumed by A. tumefaciens as carbon and nitrogen sources. Outside the T-DNA are located the
genes for the opine catabolism, the genes involved in the process of TDNA transfer from the
bacterium to the plant cell and the genes involved in bacterium-bacterium plasmid conjugative
transfer (Hooykaas and Schilperoort, 1992; Zupan and Zambrysky, 1995).
Fig : structure of Ti plasmid
Virulent strains of A. tumefaciens and A. rhizogenes, when interacting with susceptible
dicotyledonous plant cells, induce diseases known as crow gall and hairy roots, respectively.
These strains contain a large megaplasmid (more than 200 kb) which play a key role in tumor
induction and for this reason it was named Ti plasmid, or Ri in the case of A. rhizogenes. Ti
plasmids are classified according to the opines, which are produced and excreted by the tumors
they induce. During infection the T-DNA, a mobile segment of Ti or Ri plasmid, is transferred to
the plant cell nucleus and integrated into the plant chromosome. The T-DNA fragment is flanked
by 25-bp direct repeats, which act as a cis element signal for the transfer apparatus. The process
of T-DNA transfer is mediated by the cooperative action of proteins encoded by genes
determined in the Ti plasmid virulence region (vir genes) and in the bacterial chromosome. The
Ti plasmid also contains the genes for opine catabolism produced by the crown gall cells, and
regions for conjugative transfer and for its own integrity and stability. The 30 kb virulence (vir)
region is a regulon organized in six operons that are essential for the T-DNA transfer (virA, virB,
virD, and virG) or for the increasing of transfer efficiency (virC and virE) (Hooykaas and
Schilperoort, 1992; Zupan and Zambryski, 1995, Jeon et al., 1998). Different chromosomal-
determined genetic elements have shown their functional role in the attachment of A. tumefaciens
to the plant cell and bacterial colonization: the loci chvA and chvB, involved in the synthesis and
excretion of the b-1,2 glucan (Cangelosi et al., 1989); the chvE required for the sugar
enhancement of vir genes induction and bacterial chemotaxis (Ankenbauer et al., 1990,
Cangelosi et al., 1990, 1991); the cel locus, responsible for the synthesis of cellulose fibrils
(Matthysse 1983); the pscA (exoC) locus, playing its role in the synthesis of both cyclic glucan
and acid succinoglycan (Cangelosi et at., 1987, 1991); and the at the locus, which is involved in
the cell surface proteins (Matthysse, 1987).
The initial results of the studies on T-DNA transfer process to plant cells demonstrate three
important facts for the practical use of this process in plants transformation. Firstly, the tumor
formation is a transformation process of plant cells resulted from transfer and integration of T-
DNA and the subsequent expression of T-DNA genes. Secondly, the T-DNA genes are
transcribed only in plant cells and do not play any role during the transfer process. Thirdly, any
foreign DNA placed between the T-DNA borders can transferred to plant cells, no matter where
it comes from. These well-established facts, allowed the construction of the first vector and
bacterial strain systems for plant transformation (for review Hooykaas and Schilperoort, 1992;
Deblaere et al., 1985; Hamilton, 1997; Torisky et al., 1997).
The first record on transgenic tobacco plant expressing foreign genes appeared at the beginning
of the last decade, although many of the molecular characteristics of this process were unknown
at that moment (Herrera-Estrella, 1983). Since that crucial moment in the development of plant
science, a great progress in understanding the Agrobacterium-mediated gene transfer to plant
cells has been archived. However, Agrobacterium tumefaciens naturally infects only
dicotyledonous plants and many economically important plants, including the cereals, remained
accessible for genetic manipulation during long time. For these cases, alternative direct
transformation methods have been developed (Shillito et al, 1985; Potrykus, 1991) such as
polyethyleneglycol-mediated transfer (Uchimiya et al., 1986), microinjection (de la Pena et al.,
1987), protoplast and intact cell electroporation (Fromm et al., 1985, 1986; Lörz et al., 1985;
Arencibia 1995) and gene gun technology (Sanford, 1988). However, Agrobacterium-mediated
transformation has remarkable advantages over direct transformation methods. It reduces the
copy number of the transgene, potentially leading to fewer problems with transgene
cosuppresion and instability (Koncz et al., 1994, Hansen et al., 1997). In addition, it is a single-
cell transformation system not forming mosaic plants, which are more frequent when direct
transformationis used (Enríquez-Obregón et al 1997, 1998).
Fig : Intergration of Agrobaterium into plant cell
Agrobacterium tumefaciens T-DNA transfer process
The process of gene transfer from Agrobacterium tumefaciens to plant cells implies several
essential steps:
(1) Bacterial colonization (2) induction of bacterial virulence system, (3) generation of T-DNA
transfer complex (4) TDNA transfer and (5) integration of T-DNA into plant genome. A
hypothetical model depicting the most important stages of this process is presented, supported by
the most recent experimental data and accepted hypothesis on T-DNA transfer.
Figure: Basic steps in transformation of plant cells by Agrobacterium tumefaciens. The T-DNA
transfer is represented according to updated knowledge on this process, although many of
involved mechanisms have not been elucidated yet and the experimental results only allow
hypothesize about it. Entering of T-DNA complex into the plant cell is almost completely
uncharacterized and experimentally only the VirB7-VirB9 disulfide bound heterodimers have
been evidenced.The most important events are briefly mentioned in chronological order (boxes 1
through 13).
Fig: virulence genes of T-DNA
Bacterial colonization: Bacterial colonization is an essential and the earliest step in tumor
induction and it takes place when A. tumefaciens is attached to the plant cell surface (Matthysse ,
1986). Mutagenesis studies show that non-attaching mutants loss the tumor-inducing capacity
(Cangelosi et al., 1987, Douglas et al., 1982, Thomashow et al., 1987, Bradley et al., 1997). The
polysaccharides of the A. tumefaciens cell surface are proposed to play an important role in the
colonizing process. The bacterial attachment could be prevented when lipopolysaccharides (LPS)
solution from virulent strains is applied to the plant tissue before interaction with virulent
bacteria (Whatley and Spiess, 1977). A. tumefaciens, like other plant-associative Rhizobiaceae
bacteria, produces also capsular polysaccharides (Kantigens) lacking lipid anchor and having
strong anionic nature and tight association with the cell.
Fig: Agrobacterium tumefaciens colonizing tobacco leaves
The chromosomal 20kb att locus contains the genes required for successful bacterium attachment
to the plant cell. This locus has been extensively studied using transposon insertion mutants.
Insertions in the left 10 kb side of this region produced avirulent mutants that could restore its
attachment capacity if the culture medium was previously conditioned by the incubation of wild-
type virulent bacterium with plant cells.
Induction of bacterial virulence system: The T-DNA transfer is mediated by products
encoded by the 30-40 kb vir region of the Ti plasmid. This region is composed by at least six
essential operons (vir A, vir B, vir C, vir D, vir E, virG ) and two non-essential (virF, virH). The
number of genes per operon differs, virA, virG and virF have only one gene; virE, virC, virH
have two genes while virD and virB have four and eleven genes respectively. The only
constitutive operons are virA and virG, coding for a two-component (VirA-VirG) system
activating the transcription of the other vir genes. The VirA-VirG two-component system has
structural and functional similarities to other already described for other cellular mechanisms
(Nixon, 1986, Iuchi, 1993).
VirA is a transmembrane dimeric sensor protein that detects signal molecules, mainly small
phenolic compounds, released from wounded plants (Pan et al., 1993). The signals for VirA
activation include acidic pH, phenolic compounds, such as acetosyringone (Winans et al., 1992),
and certain class of monosaccharides which acts sinergistically with phenolic compounds
(Ankenbauer et al., 1990; Cangelosi et al., 1990; Shimoda et al., 1990; Doty et al., 1996). VirA
protein can be structurally defined into three domains: the periplasmic or input domain and two
transmembrane domains (TM1 and TM2). The TM1 and TM2 domains act as a transmitter
(signaling) and receiver (sensor) (Parkinson, 1993). The periplasmic domain is important for
monosaccharide detection (Chang and Winans, 1992). Within the periplasmic domain, adjacent
to the TM2 domain is an amphipatic helix, with strong hydrophilic and hydrophobic regions
(Heath et al., 1995). This structure is characteristic for other transmembrane sensor proteins and
folds the protein to be simultaneously aligned with the inner membrane and anchored in the
membrane (Seligman and Manoil, 1994). The TM2 is the kinase domain and plays a crucial role
in the activation of VirA, phosphorylating itself on a conserved His-474 residue (Huang et al.,
1990; Jin et al. 1990a, 1990b) in response to signaling molecules from wounded plant sites.
Monosaccharide detection by VirA is an important amplification system and responds to low
levels of phenolic compounds. The induction of this system is only possible through the
periplasmic sugar (glucose/galactose) binding protein ChvE (Ankenbauer and Nester, 1990;
Cangelosi et al., 1990), which interacts with VirA (Shimoda et al., 1990, 1993; Turk et al., 1993;
Chang and Winans, 1992). Recent studies for determination of VirA regions, important for its
sensing activity suggested the position, which may be involved on TM1-TM2 interaction . This
interaction causes the exposure of the amphipathic helix to small phenolic compounds and
suggests a putative model for the VirAChvE interaction (Doty et al., 1996).
Activated VirA has the capacity to transfer its phosphate to a conserved aspartate residue of the
cytoplasmic DNA binding protein VirG (Jin et al. 1990a, 1990b; Pan et al., 1993). VirG
functions as a transcriptional factor regulating the expression of vir genes when it is
phosphorilated by VirA (Jin et al., 1990a, 1990b). The C-terminal region is responsible for the
DNA binding activity, while the N terminal is the phosphorylation domain and shows homology
with the VirA receiver (sensor) domain.
The activation of vir system also depends on external factors like temperature and pH. At
temperatures greater than 32°C, the vir genes are not expressed because of a conformational
change in the folding of VirA induce the inactivation of its properties. (Jin et al., 1993). The
effect of temperature on VirA is suppressed by a mutant form of VirG (VirGc), which activates
the constitutive expression of the vir genes. Hovewer, this mutant cannot confers the virulence
capacity at that temperature to Agrobacterium, probably because the folding of other proteins
that actively participate in the T-DNA transfer process are also affected at high temperature
(Fullner and Nester, 1996).
Generation of T-DNA transfer complex: The activation of vir genes produces the generation
of single-stranded (ss) molecules representing the copy of the bottom T-DNA strand. Any DNA
placed between T-DNA borders will be transferred to the plant cell as single strand DNA and
integrated into the plant genome. These are the only cis acting elements of the T-DNA transfer
system. The proteins VirD1 and VirD2 play a key role in this step, recognizing the T-DNA
border sequences and nicking (endonuclease activity) the bottom strand at each border. The nick
sites are assumed as the initiation and termination sites for T-strand recovery. After
endonucleotidic cleavage, VirD2 remains covalently attached to the 5’-end of the ssT-strand.
This association prevents the exonucleolytic attack to the 5’-end of the ss-T-strand (Dürrenberger
et al., 1989) and distinguishes the 5’-end as the leading end of the T-DNA transfer complex.
VirD1 interacts with the region where the ss-T-strand is originated. Experiments in vitro
evidenced that the presence of VirD1 is essential for thecleavage of supercoiled stranded
substrate by VirD2 (Zupan and Zambryski, 1995; Christie et al., 1997). The simultaneous
restoration of the excised ssT-strand is evolutionarily related to other bacterial conjugative DNA
transfer processes, which includes the generation of the single strand DNA (Zupan and
Zambryski, 1995; Christie et al., 1997; Lessl et al., 1994).
Integration of T-DNA into plant genome: Inside the plant cell, the ssT-DNA complex is
targeted to the nucleus crossing the nuclear membrane. Two Vir proteins have been found to
be important in this step: VirD2 and VirE2, which are the most important, and probably
VirF, which has a minor contribution to this process (Hooykaas and Schilperoort, 1992). The
nuclear location signals (NLS) of VirD2 and VirE2 play an important role in nuclear
targeting of the delivered ss-TDNA complex, as early described. VirD2 has one functional
NLS. The ssT-DNA complex is a large (up to 20 kb) nucleoprotein complex containing only
one 5’end covalently attached VirD2 protein per complex. But the complex is coated by a
large number of VirE2 molecules (approximately 600 per a 20 kb T-DNA), and each of them
has two NLS. The two NLS of VirE2 have been considered important for the continuos
nuclear import of ss-T-DNA complex, probably by keeping both sides of nuclear pores
simultaneously open. The nuclear import is probably mediated also by specific NLS-binding
proteins, which are present in plant cytoplasm.
The final step of T-DNA transfer is its integration into the plant genome. The mechanism
involved in the T-DNA integration has not been characterized. It is considered that the
integration occurs by illegitimate recombination (Gheysen et al., 1991, Lehman et al., 1994;
Puchta, 1998). According to this model, paring of a few bases, known as micro-homologies,
are required for a pre-annealing step between T-DNA strand coupled with VirD2 and plant
DNA. These homologies are very low and provide just a minimum specificity for the
recombination process by positioning VirD2 for the ligation. The 3´-end or adjacent
sequences of T-DNA find some low homologies with plant DNA resulting in the first contact
(synapses) between the T-strand and plant DNA and forming a gap in 3’-5’ strand of plant
DNA. Displaced plant DNA is subsequently cut at the 3’-end position of the gap by
endonucleases, and the first nucleotide of the 5’ attaches to VirD2 pairs with a nucleotide in
the top (5’-3’) plant DNA strand. The 3’ overhanging part of T-DNA together with displaced
plant DNA are digested away, either by endonucleases or by 3’- 5’ exonucleases. Then, the
5’ attached to VirD2 end and other 3’-end of T-strand (paired with plant DNA during since
the first step of integration process) joins the nicks in the bottom plant DNA strand. Once the
introduction of T strand in the 3’-5’ strand of the plant DNA is completed, a torsion followed
by a nick into the opposite plant DNA strand is produced. This situation activates the repair
mechanism of the plant cell and the complementary strand is synthesized using the early
inserted T-DNA strand as a template (Tinland et al., 1995).
Agrobacterium-mediated transformation of sugarcane (Saccharum oficinarum L.)
Sugarcane (Saccharum officinarum L.) is cultivated on large scale in tropical and subtropical
regions as raw material for sugar and industrial products, such as furfural, dextrans, and alcohol
(Martín et al., 1982). Sugarcane represents 65 % of the world sugar production (Agra -Europe
1995). Traditional plant breeding techniques, together with classic biotechnological approaches,
have been extensively used to increase crop yields by selecting improved varieties which are
more productive and resistant to diseases and pathogens.
The use of plant transformation methods to introduce resistance genes into plant genomes may
have an important impact on sugarcane yields. Recently, our group published a report evidencing
the generation of the first transgenic sugarcane lines resistant to stem-borer attack (Arencibia et
al. 1997).
The lack of a reproducible methodology for stable transformation of sugarcane was an important
obstacle for its genetic manipulation during many years. In 1992, Bower and Birch successfully
recovered transgenic sugarcane plants from cell suspensions and embryogenic calli transformed
by particle bombardment (Bower and Birch, 1992). Simultaneously, Arencibia et al. (1992)
developed a procedure for stable transformation of sugarcane by electroporation of meristematic
tissue. Later, a method to produce transgenic sugarcane plants by intact cell electroporation was
established by the same group (Arencibia et al. 1995). The development of herbicideresistant
plants containing the bar gene and derived from the commercial variety NCo 310 by biolistic
transformation (Gallo-Meagher and Irvine 1996) has been recently reported. However, direct
plant-transformation systems are known to be traumatic to the cells, expensive due to the need of
special equipment, and poorly reproducible because of the variable transgene copy-number per
genome.
Agrobacterium versus particle bombardment
In general Agrobacterium is considered the method of choice for transformation.
Advantages include:
low copy number of the transgene
higher proportion of stable transformants
larger DNA segments can be transferred
more time-efficient
Are there alternatives?
Electroporation of plant protoplasts/pollen/embryos/callus. In this case the DNA is
introduced while the cells are subjected to an electric discharge.
Micro-injection. The DNA is injected into the protoplast with a pipet
Silicon-carbide mediated transformation. DNA and silicon carbide fibers are mixed in
with cells and mixed in a blender. The DNA-coated fibers penetrate the cells via small
holes created by the fibers and introduce the DNA.
Pollen-tube pathway. DNA can be applied to cut styles shortly after pollination and
flows down the pollen tube to end up in the zygote.
Liposomes. DNA is loaded into phospholipid spheres (liposomes) and these liposomes
are mixed with protoplasts to result in lipofection
These alternatives are typically not very efficient….
METHODOLOGY
Transient and Stable Transfection
Transient transfection: In transient transfection, the transfected DNA is not integrated into host
chromosome. DNA is transferred into a recipient cell in order to obtain a temporary but high
level of expression of the target gene.
Stable transfection: Stable transfection is also called permanent transfection. By the stable
transfection, the transferred DNA is integrated (inserted) into chromosomal DNA and the
genetics of recipient cells is permanent changed.
Transfection Methods
Generally, there are 9 ways for gene transfer: (1) Lipid-mediated method; (2) Calcium-phosphate
mediated; (3) DEAE-dextran-mediated; (4) Electroporation; (5) Biolistics; (6) Viral vectors; (7)
Polybrene; (8) Laser transfection; (9) Gene transfection enhanced by elevated temperature
(Sambrook, 2001).
1. Lipid-mediated method: This method can be used for both transient and stable
transfection, and it can be used for adherent cells, primary cell lines, and suspension cultures. For
the following protocol, the Lipofectamine Reagent from Invitrogen Corporation will be used.
Lipofectamine Reagent is a 3:1 (w/w) liposome formulation of the plycationic lipid 2,3-
dioleyloxy-N-[2(sperminecardoxido)ethyl]-N,N-dimethyl-1-propanaminium trifluoro acetate
(DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane-filtered
water (Catalogue Number 18324, Invitrogen Corporation, Carlsbad, California, USA) (Hawley-
Nelson, 1993; Shih, 1997).
(1) Put about 40,000 cells per well of a 24-well plate in 0.5 ml of the appropriate complete
growth medium (add 10% serum if it needs).
(2) Incubate the cells at 37oC in a CO2 incubator until the cells are 50-80% confluent (about 20
hours, depending on the cells).
(3) Dilute 3 μg DNA into 25 μl medium without serum for each well and mix.
(4) Dilute 3 μl Lipofectamine Reagent into 25 μl medium without serum for each well and mix.
(5) Combine diluted DNA (Step 3) and Lipofectamine Reagent (Step 4) and incubate at room
temperature for 30 min. In this step the DNA-liposome complexes are formed.
(6) Replace the medium in the cells with 0.2 ml transfection medium without serum.
(7) Add 0.15 ml medium without serum to the tube containing the complexes for each well.
(8) Incubate the cells with the complexes for about 10 hours at 37oC in a CO2 incubator. The
incubating time will be flexible by the cell type.
(9) Add 0.4 ml growth medium containing double the 2× normal concentration of the serum
without removing the transfection mixture.
(10) Replace the medium with fresh, complete medium at 20 hours following the start of
transfection if continued cell growth is required.
(11) Assay cell extracts for transient gene expression at 24-72 hours after transfection, depending
on the cell type and promoter activity.
(12) To obtain stable transfectants, passage the cells 1:10 into the selective medium after 72
hours of transfection for the reporter gene transfected.
2. Calcium-phosphate mediated: To get a better description, the following protocol is using the
human interleukin-2 gene transfer into cultured rat myocytes as the example manual.
2.1 Rat heart muscle cells are primarily cultured:
(1) Adult rats are sacrificed by decapitation with a decapitator.
(2) Rat hearts are moved out and left atria are isolated under sterile condition.
(3) Tissue is transferred to a fresh sterile phosphate buffered solution (PBS) and rinse.
(4) Transfer to a second dish and dissect off unwanted tissue such as fat or necrotic material and
chop finely with crossed scalpels to about 1 mm cubes.
(5) Transfer by pipette (10 – 20 ml with wide tip) to a 15-ml sterile centrifuge tube.
(6) Wash by resuspending the pieces in PBS, transfer the chopped pieces to the trypsinization
flask, and add 1 ml trypsin solution (0.25%) per 100 mg tissue. Incubate the tissue in trypsin
solution for 12 hours at 4oC then wash with PBS for 3 times.
(7) Add 1 ml trypsin solution (0.25%) per 100 mg tissue, with 1 mg/ml elastase and 1 mg/ml
collagenase then stir at about 200 rpm for 30 min at 36.5 oC.
(8) Allowing the pieces to settle, collect supernatant, centrifuge at approximately 500 g for 5
min, resuspending pellet in 10 ml medium with 10% serum (FBS), and store cells on ice.
(9) Add fresh trypsin to pieces and continue to stir and incubate for a further 30 min. Repeat
steps 6 – 8 until complete disaggregation occurs or until no further disaggregation is apparent.
(10) Collect and pool chilled cell suspensions, and count by hemocytometer.
(11) Dilute to 106 per ml in growth medium and seed as many flasks as are required with
approximately 2 x 105 cells per ml or set up a range of concentrations from about 10 mg tissue
per ml.
(12) Put into CO2 incubator with 36.5oC.
(13) Culture medium used is Medium 199 with 10% FBS. All the solutions used contain 0.1
mg/ml of anti-biotic ampicillin .
2.2 Bacteria Culture (Sambrook, 1989; Frederick, 1992):
(1) Growth of E. coli:
Dissolve E. coli in 0.3 ml LB plus tetracycline (2 mg/ml) medium, transfer it into a tube
containing 5 ml LB plus tetracycline (2 mg/ml) medium, 37oC overnight, then freeze it at -70oC.
(2) Harvesting E. coli:
A. Streak an inoculum across one side of a plate. Resterilize an inoculating loop and streak a
sample from the first streak across a fresh part of plate, then incubate at 37oC until colonies
appear (overnight).
B. Transfer a single bacterial colony into 2 ml of LB medium containing tetracycline (2 mg/ml)
in a loosely capped 15-ml tube. 37oC overnight with vigorous shaking.
C. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at 12,000g for 30 seconds at 4oC
in a microfuge. Store remainder at 4 oC.
D. Remove the medium by aspiration.
(3) Lysis of E. coli and purification of plasmid:
A. Resuspend E. coli pellet in 100 μl of ice-cold Solution I (50 mM glucose, 25 mM Tris-Cl, pH
8.0, 10 mM EDTA, pH 8.0).
B. Add 200 μl of freshly prepared Solution II (0.2 N NaOH, 1% SDS), inverting the tube ra pidly
5 times. Do not vortex. Store at 4oC.
C. Add 150 μl ice-cold Solution III (5 M potassium acetate 60 ml, glacial acetic acid 11.5 ml,
H2O 28.5 ml), on ice for 3-5 min.
D. Centrifuge at 12,000g for 10 min, at 23oC .
E. Pour supernatant into QIAprep column (silicon gel column, Qiagen Company, USA).
F. Centrifuge at 12000g for 1 min and discard flow through.
G. Wash the column with 0.75 ml PE buffer (55 ml of 5 mM Mops-KOH, pH 7.5-7, 0.75 mM
NaCl plus 220 ml of ethanol).
H. Centrifuge 1 min at 12000g and discard flow through.
I. Place column in 1.5 ml microcentrifuge tube.
J. Add 50 μl of the DEPC H2O in the center of the column, stand for 1 min, centrifuge at 12000g
for 1 min.
K. Take 1 μl of DNA (plasmid), add 99 μl of TE buffer, pH 8.0, measure DNA concentration at
OD260 nm and OD280 nm (OD260 nm/OD280 nm should be >1.7).
L. Redissolve the DNA in 50 μl of TE (pH 8.0) containing DNAase-free pancreatic RNAase (20
μg/ml). Vortex briefly. Store at -20oC.
M. Calculate the concentration of the plasmid DNA: 1 OD260 nm = 50 μg of plasmid DNA/ml.
Store the DNA in aliquots at -20oC.
2.3 Transfer human interleukin-2 gene into rat heart muscle cells:
(1) Transferred gene: Human interleukin-2 (IL-2) gene cloned in plasmid pBR322 inserted in E.
coli can be bought from American Type Culture Collection.
(2) Transfection: ∼2×107 of heart muscle cells suspended in 0.2 ml medium are seeded into a
tissue culture chamber. 48-72 hours later, remove medium and add 0.2 ml fresh medium, then
add 0.5 μg of plasmid in 0.05 ml calcium phosphate-HEPES-buffered saline, pH 7.0, at 37 oC.
3 DEAE-dextran mediated : DEAE-dextran (diethylaminoethyloethyl-dextran) was used to
introduce poliovirus RNA and SV40 and polyomavirus DNAs into cells in 1960s (Pagano, 1965;
McCutchan, 1968; Warden, 1968).
There are three points that DEAE-dextran mediated transfection differs from calcium phosphate
coprecipitation. (1) It is used for transient transfection. (2) It works more efficiently with cell
lines of BSC-1, CV-1 and COS, etc. (3) It is more sensitive.
The DEAE-dextran mediated transfection could be done by the following steps:
(1) Harvest exponentially growing cells by trypsinization and transfer then into 60 -mm tissue
culture dished at a density of 105 cells/dish.
(2) Add 5 ml complete growth medium.
(3) Incubate 24 hours at 37oC with 5% CO 2.
(4) Prepare DNA/DEAE-dextran/TBS-D solution by mixing 2 mg of superoiled plasmid DNA
into 1 μg/ml DEAE-dextran in TBS-D.
(5) Remove medium and wash tree times with PBS and twice with TBS-D.
(6) Add DNA/DEAE-dextran/TBS-D solution 250 μl.
(7) Incubate 60 min at 37oC with 5% CO 2.
(8) Remove DNA/DEAE-dextran/TBS-D solution.
(9) Wash with TBS-D three time and PBS twice.
(10) Add 5 ml medium supplemented with serum and chloroquine (0.1 mM).
(11) Incubate 4 hours at 37oC with 5% CO2 .
(12) Remove medium.
(13)Wash with serum-free medium three times.
(14) Add to cells 5 ml of medium supplement with serum, and incubate 48 hours at 37 oC with
5% CO2.
(15) Harvest the cells after the 48 hours transferction.
(16) Analyze RNA or DNA by hybridization, or analyze expressed protein by radiommunoassay,
immunoblotting, immuniprecipitation, or by enxzymoatic activity in cell extract.
4 Electroporation: Pulse electrical fields can be used to introduce DNA into cells of animal,
plant and bacteria. Factors that influence efficiency of transfection by electroporation: applied
electric field strength, electric pulse length, temperature, DNA conformation, DNA
concentration, and ionic composition of transfection medium, etc.
Steps of the electroporation transfection:
(1) Harvest cells in the mid- to late-logarithmic phase of growth.
(2) Centrifuge at 500 g (2000 rpm) for 5 min at 4oC.
(3) Resuspend cells in growth medium at concen-
tration of 1 X 107 cells/ml.
(4) Add 20 μg plasmid DNA in 40 μl cells.
(5) Electric transfect by 300 V / 1050 μF for 1-2 min.
(6) Transfer the electroporated cells to culture dish and culture the cells.
(7) Assay DNA, RNA or protein and continuously culture the cells to get positive cell lines. .
5 Polybrene:
Several polycations, including polybrene (1,5-dimethyl-1,5-diazaundecamethylene poly-
methobromide) (Chaney, 1986) and poly-L-ornithine (Nead, 1995), have been used in gene
transfection with the DMSO enhancement. Normal steps are following:
(1) Harvest exponential cells by trypsinzationin and replate at a density of 5,000 cells/mm2 in 10
ml MEM-α containing 10% fetal calf serum.
(2) Incubate 24 hours at 37oC in 5% CO2.
(3) Replace medium with 3 ml pre-warmed medium containing serum, 10 μg DNA and 30 μg
polybrene (37oC). Mix the medium before adding polybrene.
(4) Incubate 12 hours with a gent shake each hour.
(5) Remove medium and add 5 ml 30%^ DMSO in serum-containing medium.
(6) After 4 min incubation, aspirate the DMSO solution. Wash the cells twice with warmed
(37oC) serum-free medium, and add 10 ml complete medium containing 10% fetal calf serum.
(7) Incubate 48 hours at 37oC in 5% CO2.
(8) Examine the cells everyday after the transferction.
(9) For stable transfection, continue incubate 3 weeks with changing medium every 2 days.
6 Virus: Viruses are highly adapted to the process of gene transfer. Viral vectors have the ability
to transfer DNA to a high fraction of cells, but using virus as the vector will be potentially arouse
cancer leukaemia (Cavazzana-Calvo, 2004). Common vectors used for gene transfer in cell
culture are derived from retroviruses. Adenovirus and other agents are used for the gene delivery.
7 Biolistics (Gene gun, or called microparticle bombardment) : Some cells, tissues and
intracellular organelles are impermeable to foreign DNA, especially plant cells. biolistics,
including particle bombardment, is a commonly used method for genetic transformation of plants
and other organisms. To resolve this problem in gene transfer, the gene gun was made by Klein
at Cornell University in 1987 (Klein, 1987; Kikkert, 2005). On the gene gun technique, Klein
and Sanford et al published papers, obtained patents and formed a company called Biolistics
(Klein, 1987).
The gene gun is part of the gene transfer method called the biolistic (also known as biobalistic or
particle bombardment) method. In this method, DNA or RNA adhere to biological inert particles
(such as gold or tungsten). By this method, DNA-particle complex is put on the top location of
target tissue in a vacuum condition and accelerated by powerful shot to the tissue, then DNA will
be effectively introduce into the target cells. Uncoated metal particles could also be shot through
a solution containing DNA surrounding the cell thus picking up the genetic material and
proceeding into the living cells. The efficiency of the gene gun transfer could be depended on the
following factors: cell type, cell growth condition, culture medium, gene gun ammunition type,
gene gun settings and the experimental experiences, etc.
Briefly for gene gun practice, the target cells or tissues on the polycarbonate membranes could
be positioned in a Biolistic PDS-1000/HE Particle Delivery System (Bio-Rad Laboratories
GmbH, München, Germany). Biolistic parameters are 15 in. Hg of chamber vacuum, target
distance of 3 cm (stage 1), 900 psi to 1800 psi particle acceleration pressure, and 1.0 μm
diameter gold microcarriers (Bio-Rad, USA). Gold microcarriers are prepared, and circular
plasmid DNA is precipitated onto the gold using methods recommended by Bio-Rad with the
following: 0.6 mg of gold particles carrying ~5 μg of plasmid DNA is used per bombardment.
The detail protocol for the gene gun transfection is described as follows:
(1) Prepare gold or tungsten particles: 60 mg gold or tungsten in 1 ml 70% ethanol, centrifuge at
10,000 rpm for 10 seconds and collect particles, and wash with H2O three times by
centrifugation.
(2) Prepare DNA-coated particles: Mix 50 l (about 3 mg) metal, 2.5 l plasmid DNA (about 2.5 -
g), CaCl2 50 l (2.5 M), spermidine 20 l (0.1M). Vortex and stand for 5 min. Centrifuge, remove
supernatant, and add 140 l 70% ethanol over the pelleted particles, and repeat the ethanol and
centrifugation three times, then add 50 l ethanol.
(3) Place a macrocarrier in the metal holder of gene gun and wash twice with ethanol.
(4) Vortex and spread 0.5 mg pellet slurry on the macrocarrier.
(5) Load the macrocarrier into the gene gun, and shoot it. Repeat the shoot until all the areas are
shot. For transient expression, examine cells 48 hours after the shooting, by immunology or other
methods. For stable transfection, continue culture the transfected cells or tissues.
8 Laser transfection: As the examples, UV excimer laser (XeCl2, 308 nm) is used in the gene
transfection (5 min by a 0.7 0.9, 1.4 or 2.0 mm diameter fiber with fluence of 45 and 60 mj/mm2
- real laser energy 2.3, 5.9, 13.1, 32.0 mj/pulse, 25 Hz) (CVX-300 Excimer Laser System,
Spectranetics Corporation, Colorado Springs, CO, USA). Also, we used to make experiments
with Nd:Yag, Ho:Yag in the gene transfection. All the methods of excimer, Nd:Yag and Ho:Yag
laser transfection are effective.
9 Transfection enhanced by elevated temperature: Studies shows that high temperature
enhances the gene transfection. In our experiments, rat heart muscle cells were cultured in
medium 199 with 10% FBS and human aorta smooth muscle cells were cultured in F12K
medium. Human interleukin-2 gene was transfected into rat heart cells and swine growth
hormone gene was transfected into human aorta smooth muscle cells by calcium phosphate
coprecipitation at various temperatures: 23ºC, 37ºC and 43ºC. Transfected interleukin-2 and
swine growth hormone expressions were detected using an indirect ELISA. The heated cultured
rat myocytes had a significantly higher expression of the transfected interleukin-2 gene. Ambient
temperature rise to 43oC for up to 30 min provided greater transient transfection of the
interleukin-2 gene when compared to ambient temperatures at 37oC and 23oC (p<0.01). The
greatest effects occurred within 10 min of incubation and persisted up to 30 min. These results
suggest that even a few degrees of ambient temperature rise can significantly increase gene
transfer into muscle cells. This may be of value when using gene therapy with transfection
procedures (Ma, 2004b; Ma, 2004c).
10 Plant gene transfer: Agriculture and plant breeding relied solely on the accumulated
experience of generations of farmers and breeders that is, on sexual transfer of genes between
plant species. However, developments of plant molecular biology and genomics now give us
access to knowledge and understanding of plant genomes and the possibility of modifying them.
There are two most powerful technologies for transferring gene into plants: Agrobacterium-
mediated transformation and biolistics. As plants have cell wall, the biolistics is very useful in
the plant gene transfer (Rasco-Gaunt, 2001).
DISCUSSION & CONCLUSION
The current century will bring tremendous changes to the science, technology, and the practice of
medicine (Lushai, 2002). Gene therapy is part of a growing field in molecular medicine, which
will gain importance in the treatment of human diseases (Gunther, 2005). As a critical topic,
gene transfection gives people the hope to treat many diseases but it also could create dangerous
species in the earth, so that it attracts plenty attention by the whole human society (Lanza, 2002).
This simply means that the success of gene transfer technique will be benefit for the civilization,
and also create the danger for the life in the earth either (Schiemann, 2003). Gene transfection
procedures are used in the critic procedure animal clone (Chesne, 2002; Heyman, 2002), and the
animal clone is challenged by the religious groups and ethnic extremists (Houdebine, 2003). As
our personal views, no matter how big challenges from whatever aspects, the gene transfection
and animal clone will develop quickly. The world is a complex place composed by different
people. For the science and technology such as gene transfer and animal clone, no country can
prevent other countries from the pursuing. We need to develop the technique even if the
technique could be used in the danger action, and we need to consider the social effects of a
technique when we develop it either.
Agrobacterium tumefaciens is more than the causative agent of crown gall disease affecting
dicotyledonous plants. It is also the natural instance for the introduction of foreign genes in
plants allowing its genetic manipulation. Similarities have been found between T-DNA and
conjugal transfer systems. They are evolutionary related and apparently evolved from a common
ancestor.
Although the gene transfer mechanisms remain largely unknown, great progress has been
obtained in the implementation of transformation protocols for both dicotyledonous and
monocotyledonous plants. Particularly important is the extension of this single-cell
transformation methodology to monocotyledonous plants. This advance has biological and
practical implications. Firstly, because of the advantages of A. tumefaciens-mediated gene
transfer over the direct transformation methods, which where the only way for genetic
manipulation of economically important crops as cereals and legumes. Secondly, it has been
demonstrated that T-DNA is transferred to dicotyledonous and monocotyledonous plants by an
identical molecular mechanism. This confirmation implies that any plant can potentially be
transformed by this method if a suitable transformation protocol is established.
The Agrobacterium-mediated transformation protocols differ from one plant species to other and,
within species, from one cultivar to other. In consequence, the optimization of Agrobacterium-
mediated transformation methodologies requires the consideration of several factors that can be
determined in the successful transformation of one species. Firstly, the optimization of
Agrobacterium-plant interaction on competent cells from different regenerable tissues. Secondly,
the development of a suitable method for regeneration from transformed cells.
Undoubtedly, the development of transformation procedures based on A. tumefaciens-mediated
gene transfer for new economically important species are advisable and the results obtained in
recent years evidence a promising future.
Science development will be benefit to all the human society. As the gene therapy developing,
many more desperate diseases could be cured and many human livings could be saved, such as
the life of Pope John Paul II and Terri Schindler-Schiavo. Hope that the gene transfer techniques
described in this article could be useful for the researches in the gene therapy field and help to
advance the life science study.
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