1. Seyed Mohammad Motevalli
October 2013
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2. Outline
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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
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(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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8. Methods of creation of transgenic
animals
 DNA microinjection
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9. Methods of creation of transgenic
animals
 DNA microinjection
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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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16. How can we get embryonic cells?
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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
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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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