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Molecular Basis for Relationship between Genotype and Phenotype genotype DNA DNA sequence transcription RNA translation protein function phenotype organism amino acid sequence Method of Introducing Transgene in Mouse Refer to Figure 10-28, Griffiths et al., 2015. Determining Gene Function 1.Isolate normal gene DNA. 1.Generate mutant allele*. 1.Introduce mutant DNA* into host. 1.Score phenotype. * knockout allele knockout mutation: thymidine kinase gene Add tk+ gene. Insert neoR into exon 2. neomycin resistance gene disruption of a coding sequence Herpes of a gene by thymidine insertionkinase of a gene selectable marker. Resulting insertional mutation generates a knockout mutant allele of a gene. Targeting vector is created by adding another selectable marker. normal DNA sequence including complete gene Refer to Figure 10-29, Griffiths et al., 2015. 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 et al., 2015. 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 et al., 2015. Production of ES Cells with Gene Knockout tk+ targeting vector neoR transformation targeted insertion allele replacement ES Cell Embryonic Stem Cells: • Embryoblast or inner cell mass of blastocyst • Pluripotent • Potential for replication indefinitely Refer to Figure 10-29, Griffiths et al., 2015. Herpes thymidine kinase gene 2. ES cells are transformed with vector to knockout the normal DNA sequence. Refer to Figure 10-29, Griffiths et al., 2015. 1. ES cells contain normal DNA sequence for the gene. Herpes thymidine kinase gene Refer to Figure 10-29, Griffiths et al., 2015. NO VECTOR INSERTION: tk+ not inserted – ganciclovir resistant neoR not inserted – neomycin sensitive Refer to Figure 10-29, Griffiths et al., 2015. ECTOPIC INSERTION: tk+ inserted – ganciclovir sensitive neoR inserted – neomycin resistant double crossing over Replacement of normal gene with knockout allele by homologous recombination Refer to Figure 10-29, Griffiths et al., 2015. HOMOLOGOUS RECOMBINATION: tk+ not inserted – ganciclovir resistant neoR inserted – neomycin resistant Refer to Figure 10-29, Griffiths et al., 2015. 3 possible fates = 3 different results Targeted insertion results in cells that confer neomycinresistance and do not harbor the tk+ gene (confers ganciclovir-resistance). Cells can be isolated selectively. Refer to Figure 10-29, Griffiths et al., 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. 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 et al., 2015. 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 et al., 2015. Generation of Knockout Mice Chimeric male is mated to a/a female. Refer to Figure 10-30, Griffiths et al., 2015. 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 et al., 2015. Generation of Knockout Mice Refer to Figure 10-30, Griffiths et al., 2015. 1. Progeny mice are screened for targeted insertion. 2. Phenotype(s) of mice that are homozygous for the targeted insertion are scored. Gene Therapy Germ-line Therapy vs Somatic Therapy Gene Therapy in Mammals Human Gene Therapy Approaches to treatment of diseases DNA transfer into afflicted individual