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Understanding Genotype-Phenotype Relationship Using Gene Knockout | Gene Therapy in Mammals

Learn how genotypes influence phenotypes through gene knockout techniques in the study of DNA sequences, transcription, translation, and protein functions. Explore gene therapy approaches for disease treatment.

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Understanding Genotype-Phenotype Relationship Using Gene Knockout | Gene Therapy in Mammals

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  1. Molecular Basis for Relationship between Genotype and Phenotype genotype DNA DNA sequence transcription RNA translation amino acid sequence protein function phenotype organism

  2. Method of Introducing Transgene in Mouse Refer to Figure 10-28, Griffiths etal., 2015.

  3. Determining Gene Function • Isolate normal gene DNA. • Generate mutant allele*. • Introduce mutant DNA* into host. • Score phenotype. • * knockout allele

  4. knockout mutation: disruption of a coding sequence of a gene by insertion of a selectable marker. Resulting insertional mutation generates a knockout mutant allele of a gene. Targeting vector is created by adding another selectable marker. thymidine kinase gene Herpes thymidine kinase gene Add tk+gene. Insert neoR into exon 2. neomycin resistance gene normal DNA sequence including complete gene Refer to Figure 10-29, Griffiths etal., 2015.

  5. herpes thymidine kinase gene: Confers sensitivity to ganciclovir Herpes thymidine kinase gene neomycin resistance gene: Confers resistance to neomycin and its analogs Refer to Figure 10-29, Griffiths etal., 2015.

  6. herpes thymidine kinase gene: Confers sensitivity to ganciclovir Herpes thymidine kinase gene neomycin resistance gene: Confers resistanceto neomycin and its analogs Refer to Figure 10-29, Griffiths etal., 2015.

  7. Production of ES Cells with Gene Knockout targeting vector tk+ neoR transformation targeted insertion ES Cell allele replacement

  8. Embryonic Stem Cells: • Embryoblast or inner cell mass of blastocyst • Pluripotent • Potential for replication indefinitely Herpes thymidine kinase gene Refer to Figure 10-29, Griffiths etal., 2015.

  9. 1. ES cells contain normal DNA sequence for the gene. 2.ES cells are transformed with vector to knockout the normal DNA sequence. Herpes thymidine kinase gene Refer to Figure 10-29, Griffiths etal., 2015.

  10. Refer to Figure 10-29, Griffiths etal., 2015. NO VECTOR INSERTION: tk+ not inserted – ganciclovir resistant neoR not inserted – neomycin sensitive

  11. Refer to Figure 10-29, Griffiths etal., 2015. ECTOPIC INSERTION: tk+ inserted – ganciclovir sensitive neoR inserted – neomycin resistant

  12. double crossing over Replacement of normal gene withknockout allele by homologous recombination Refer to Figure 10-29, Griffiths etal., 2015. HOMOLOGOUS RECOMBINATION: tk+ not inserted – ganciclovir resistant neoR inserted – neomycin resistant

  13. Refer to Figure 10-29, Griffiths etal., 2015. 3 possible fates = 3 different results Targeted insertion results in cells that confer neomycin-resistanceand do not harbor the tk+ gene (confers ganciclovir-resistance). Cells can be isolated selectively.

  14. Refer to Figure 10-29, Griffiths etal., 2015. In the presence of neomycin analog and ganciclovir, only cells with targeted insertion will survive. Such cells can be cultured and introduced into embryos in the blastocyst stage.

  15. Generation of Knockout Mice Embryo is inserted into a surrogate mother… agouti ES cells from agouti mouse (A/A) with targeted insertion of a mutant allele are inserted into blastocyst-stage embryo of a/a genotype. Refer to Figure 10-30, Griffiths etal., 2015.

  16. Generation of Knockout Mice Progeny that are chimeric (black and agouti) for coat color are selected. Embryo is inserted into a surrogate mother… Refer to Figure 10-30, Griffiths etal., 2015.

  17. Generation of Knockout Mice Chimeric male is mated to a/a female. Refer to Figure 10-30, Griffiths etal., 2015.

  18. Generation of Knockout Mice DNA from progeny are screened for targeted insertion. A/a mice with targeted insertion are crossed. Refer to Figure 10-30, Griffiths etal., 2015.

  19. Generation of Knockout Mice 1. Progeny mice are screened for targeted insertion. Refer to Figure 10-30, Griffiths etal., 2015. 2. Phenotype(s) of mice that are homozygous for the targeted insertion are scored.

  20. Gene Therapy Germ-line Therapy vs Somatic Therapy

  21. Gene Therapy in Mammals

  22. Human Gene Therapy • Approaches to treatment of diseases • DNA transfer into afflicted individual

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