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Genetic engineering in animal cells
This document outlines methods for creating transgenic animals. It begins with definitions and historical background, then describes the general strategy which involves isolating a gene of interest, generating a DNA construct, injecting the construct into embryos, and analyzing expression in offspring. Three main methods are discussed: DNA microinjection, retrovirus-mediated gene transfer, and embryonic stem cell-mediated gene transfer. DNA microinjection involves directly injecting DNA into the nucleus of cells, but results in transgenic progeny in less than 5% of cases. Retroviral vectors can be used to introduce genes. Embryonic stem cells are modified then injected into blastocysts. The document also covers getting embryonic cells, selecting transfected cells using genetic markers,
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Genetic engineering in animal cells
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- 2. Outline Definition Historical background General strategy Methodsof 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 2
- 3. Definition The termtransgenic 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). 3
- 4. Historical background Duringthe 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 4
- 5. General strategy Isolationand 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 5
- 6. Methods of creationof transgenic animals Transfer of ‘naked DNA’ Calcium phosphate method DEAE-dextran Encapsulation of DNA in liposome vesicles Electroporation Micro-injection 6
- 7. Methods of creationof 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 7
- 8. Methods of creationof transgenic animals DNA microinjection 8
- 9. Methods of creationof transgenic animals DNA microinjection 9
- 10. Methods of creationof transgenic animals DNA microinjection Less than 5% of the microinjected fertilized eggs become transgenic progeny 10
- 11. Methods of creationof transgenic animals Viral-assisted DNA transfer Simian virus SV40 Retrovirus-mediated gene transfer 11
- 12. Methods of creationof transgenic animals Retrovirus-mediated gene transfer Retroviral vectors can be used to create transgenic animals 12
- 13. Methods of creationof transgenic animals Retrovirus-mediated gene transfer 13
- 14. Methods of creationof 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 14
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- 16. How can weget embryonic cells? 16
- 17. Selection of transfectedcells 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 17
- 18. Selection of transfectedcells 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 18
- 19. Gene expression Transientgene expression cells are not genetically stable and soon lose their expression ability 19
- 20. Gene expression Stablegene expression DNA is integrated into the nucleus of the host cell, or efficient extrachromosomal replication system is maintained during cell division 20
Editor's Notes
- #8 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
- #12 *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
- #14 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
- #15 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
- #20 assayed from cell culture 1-3 days after uptake of DNA
- #21 ideal recombinant cells grow with doubling time of ~18 hours, is genetically stable, and secretes a recombinant protein at ~20% of total cellular protein