Chapter 062. Principles of Human Genetics (Part 10) Transgenic Mice as Models of Genetic Disease Several organisms have been studied extensively as genetic models, including Mus musculus (mouse), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode), Saccharomyces cerevisiae (baker's yeast), and Escherichia coli (colonic bacterium). The ability to use these evolutionarily distant organisms as genetic models that are relevant to human physiology reflects a surprising conservation of genetic pathways and gene function. Transgenic mouse models have been particularly valuable, because many human and mouse genes exhibit similar structure and function, and because manipulation of the mouse genome is relatively straightforward compared to those of other mammalian species. Transgenic strategies in mice can be divided into two main approaches: (1) expression of a gene by random insertion into the genome, and (2) deletion or targeted mutagenesis of a gene by homologous recombination with the native endogenous gene (knock-out, knock-in) (Fig. 62-6; Table 62-3). Transgenic mice are generated by pronuclear injection of foreign DNA into fertilized mouse oocytes and subsequent transfer into the oviduct of pseudopregnant foster mothers. Figure 62-6 Transgenic mouse models. Left. Transgenic mice are generated by pronuclear injection of foreign DNA into fertilized mouse oocytes and subsequent transfer into the oviduct of pseudopregnant foster mothers. Right. For targeted mutagenesis (gene knock-out/knock-in), embryonic stem (ES) cells are transfected with the targeted (mutagenized) transgene. The transgene undergoes homologous recombination with the wild-type gene. After selection, positive ES cells are introduced into blastocysts and implanted into foster mothers. Chimeric mice can be identified based on the mixed coat color of the offspring. Heterozygous mice are bred to obtain mice homozygous for the mutant allele. Table 62-3 Genetically Modified Animals Commonly Used Description Technical Principle Remarks Commonly used Genomic DNA or cDNA constructs Random integration of transgene Transgenic Pronuclear injection of transgene Variable copy numbers of transgene Variable expression in each individual founder Gain-of- function models due to overexpression using tissue- specific promoters Loss-of- function models using anti- sense and dominant negative transgenes Inducible expression possible (Tetracycline, ecdysone) Applicable to several species Predominantly used in mice (Targeted) Knock-out Substitution of functional gene with inactive gene by homologous Tissue-specific knock- out possible (Cre/lox) recombination in embryonic stem cells Absence of phenotype possible due to redundancy Predominantly used in mice (Targeted) Knock-in Introduction of subtle mutation(s) into gene by substitution of endogenous gene with gene carrying a specific mutation. Homologous recombination in embryonic stem cells Can accurately model human disease Selection of phenotype followed by genetic characterization Forward genetics Mutations created randomly by ENU (N- ethyl-N-nitrourea) Useful for identifying novel genes Congenic strains Mating of an inbred donor st rain with a disease phenotype with an Useful for mapping disease- causing genes inbred recipient strain in order to define the genomic region responsible for the disorder Successful in several mammalian species including sheep (Dolly), mice, cows, monkeys Cloning of genetically identical individuals May affect life-span Cloning Introduction of nucleus into enucleated eggs (nuclear transfer) Ethical concerns . Chapter 062. Principles of Human Genetics (Part 10) Transgenic Mice as Models of Genetic Disease Several organisms have been studied. Random integration of transgene Transgenic Pronuclear injection of transgene Variable copy numbers of transgene Variable expression in each individual founder Gain -of- function models. manipulation of the mouse genome is relatively straightforward compared to those of other mammalian species. Transgenic strategies in mice can be divided into two main approaches: (1) expression of