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

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

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

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 et al., 2015.

herpes thymidine kinase gene: Confers sensitivity to ganciclovir 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 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 targeting vector tk+ neoR transformation targeted insertion ES Cell allele replacement

Embryoblast or inner cell mass of blastocyst 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 et al., 2015.

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 et al., 2015.

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

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

Replacement of normal gene 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

3 possible fates = 3 different results Refer to Figure 10-29, Griffiths et al., 2015. 3 possible fates = 3 different results Targeted insertion results in cells that confer neomycin-resistance 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 1. Progeny mice are screened for targeted insertion. Refer to Figure 10-30, Griffiths et al., 2015. 2. Phenotype(s) of mice that are homozygous for the targeted insertion are scored.

Germ-line Therapy vs Somatic Therapy Gene Therapy Germ-line Therapy vs Somatic Therapy

Gene Therapy in Mammals

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