The technique of gene targeting takes advantage of the phenomenon known as homologous recombination (Fig. A.44). Cloned copies of the target gene are altered to make them nonfunctional and are then introduced into the ES cell, where they recombine with the homologous gene in the cell’s genome, replacing the normal gene with a nonfunctional copy. Homologous recombination is a rare event in mammalian cells, and thus a powerful selection strategy is required to detect those cells in which it has occurred. Most commonly, the introduced gene construct has its sequence disrupted by an inserted antibiotic-resistance gene such as that for neomycin resistance (neor). If this construct undergoes homologous recombination with the endogenous copy of the gene, the endogenous gene is disrupted but the antibiotic-resistance gene remains functional, allowing cells that have incorporated the gene to be selected in culture for resistance to the neomycin-like drug G418. However, antibiotic resistance on its own shows only that the cells have taken up and integrated the neomycin-resistance gene. To be able to select for those cells in which homologous recombination has occurred, the ends of the construct usually carry the thymidine kinase gene from the herpes simplex virus (HSV-tk). Cells that incorporate DNA randomly usually retain the entire DNA construct including HSV-tk, whereas homologous recombination between the construct and cellular DNA, the desired result, involves the exchange of homologous DNA sequences so that the nonhomologous HSV-tk genes at the ends of the construct are eliminated. Cells carrying HSV-tk are killed by the antiviral drug ganciclovir, and so cells with homologous recombinations have the unique feature of being resistant to both neomycin and ganciclovir, allowing them to be selected efficiently when these drugs are added to the cultures (see Fig. A.44).
DNA is introduced into cell Target gene interrupted by insertion of neomycin-resistance gene (neor) neorDNA fails to integrate Cell is killed by ganciclovir DNA integrates at random site on genome. Cell expresses both neorand HSV-tk Cell expresses neomycin resistance but not HSV-tk, so is not killed by either G418 or ganciclovir Homologous recombination replaces wild-type ˜2 -microglobulin gene with interrupted copy Cell is killed by G418 (a neomycin analog)
To knock out a gene in vivo, it is necessary only to disrupt one copy of the cellular gene in an ES cell. These ES cells are then injected into a blastocyst, which is reimplanted into the uterus. The cells carrying the disrupted gene become incorporated into the developing embryo and contribute to all tissues of the resulting chimeric offspring, including those of the germline. The mutated gene can therefore be transmitted to some of the offspring of the original chimera, and further breeding to homozygosity of the mutant gene produces mice that completely lack the expression of that particular gene product (Fig. A.45). The effects of the absence of the gene’s function can then be