2. Outline
Definition
Historical background
General strategy
Methods of creation of transgenic animals
DNA microinjection
Retrovirus-mediated gene transfer
Embryonic stem cell-mediated gene transfer
How can we get embryonic cells?
Selection of transfected cells
Gene expression
Transient gene expression
Stable gene expression
Transgenic Animals as Biotechnology
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3. Definition
The term transgenic animal refers to an animal in which
there has been a deliberate modification of the
genome, the material responsible for inherited
characteristics, in contrast to spontaneous mutation
(FELASA September 1992, revised February 1995).
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4. Historical background
During the 1970s, the first chimeric mice were produced
(Brinster, 1974)
DNA microinjection, the first technique to prove
successful in mammals, was first applied to mice (Gordon
and Ruddle, 1981)
retrovirus-mediated transgenesis (Jaenisch, 1976)
embryonic stem (ES) cell-mediated gene transfer (Gossler
et al., 1986)
the term transgenic was first used by J.W. Gordon and
F.H. Ruddle (1981) in 1981
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5. General strategy
Isolation and cloning of the gene of interest
Generation of a (expression-competent) DNA-construct or
genetically modified ES cells
Isolation of oocytes or blastocysts
Injection of DNA or genetically modified ES cells and transfer of
the injected embryos into a foster mouse
Analysis of the off-spring Analysis of the expression and
biological effects of the gene of interest in the transgenic animals
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6. Methods of creation of transgenic
animals
Transfer of ‘naked DNA’
Calcium phosphate method
DEAE-dextran
Encapsulation of DNA in liposome vesicles
Electroporation
Micro-injection
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7. Methods of creation of transgenic
animals
DNA microinjection
Transfection – transfer of nucleic acid into mammalian
cells (also refers to DNA-mediated transformation)
insertion of DNA directly into the nucleus of individual
cells
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10. Methods of creation of transgenic
animals
DNA microinjection
Less than 5% of the
microinjected fertilized
eggs become transgenic
progeny
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11. Methods of creation of transgenic
animals
Viral-assisted DNA transfer
Simian virus SV40
Retrovirus-mediated gene transfer
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12. Methods of creation of transgenic
animals
Retrovirus-mediated gene transfer
Retroviral vectors can be used to
create transgenic animals
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13. Methods of creation of transgenic
animals
Retrovirus-mediated gene transfer
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14. Methods of creation of transgenic
animals
Embryonic stem cell-mediated gene transfer
Embryonic stem cells
Formation of gametocytes
Injection into blastocysts
Injection into foster mother
Formation of new individual
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17. Selection of transfected cells
Cell selection by genetic markers
proportion of cells that accept exogenous DNA may
be as low as 1 in 106
marker gene incorporated into a recipient cell, allows
successful transfectants to be identified
marker can be on the same vector as target gene
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18. Selection of transfected cells
Non-dominant genes
target cells must have endogenous genes mutated or
removed, only works with mutant cells
Dominant genes
selection of any cell type, those that acquire specific
gene during transfection will survive
Antibiotic resistance genes
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19. Gene expression
Transient gene expression
cells are not genetically stable and soon
lose their expression ability
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20. Gene expression
Stable gene expression
DNA is integrated into the
nucleus of the host cell, or
efficient extrachromosomal
replication
system
is
maintained
during
cell
division
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Notes de l'éditeur
This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum
*promote transfer of DNA to cells because of capsid proteins that bind to cell membrane receptors *contain promoters that allow expression of inserted genes in animal cells * replicate to high copy numbers (several copies of required genes within cell) * some viruses’ genomic material integrate into the animal cell genome
1) Retroviruses used as vectors to transfer genetic material into the host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic constitution2) Retroviruses are also efficient vectors for integration of foreign genes into animal cellsHave RNA genome, complementary DNA copies are formed by the enzyme reverse transcriptase3) DNA can be incorporated into host cells’ genome without causing cell lysisRetroviral structural genes are replaced with foreign DNA
Genetically engineered embryonic stem (ES) cells can be used to create transgenic animals, but this method is labor intensive and used to allow for gene targeting via homologous recombination
assayed from cell culture 1-3 days after uptake of DNA
ideal recombinant cells grow with doubling time of ~18 hours, is genetically stable, and secretes a recombinant protein at ~20% of total cellular protein