Fig. A.44 The deletion of specific genes can be accomplished by homologous recombination. When pieces of DNA are introduced into cells, they can integrate into cellular DNA in two different ways. If they randomly insert into sites of DNA breaks, the whole piece is usually integrated, often in several copies. However, extrachromosomal DNA can also undergo homologous recombination with the cellular copy of the gene, in which case only the central, homologous region is incorporated into cellular DNA. Inserting a selectable marker gene such as resistance to neomycin (neor) into the coding region of a gene does not prevent homologous recombination, and it achieves two goals. First, any cell that has integrated the injected DNA is protected from the neomycin-like antibiotic G418. Second, when the gene recombines with homologous cellular DNA, the neor gene disrupts the coding sequence of the modified cellular gene. Homologous recombinants can be discriminated from random insertions if the gene encoding herpes simplex virus thymidine kinase (HSV-tk) is placed at one or both ends of the DNA construct, which is often known as a ‘targeting construct’ because it targets the cellular gene. In random DNA integrations, HSV-tk is retained. HSV-tk renders the cell sensitive to the antiviral agent ganciclovir. However, as HSV-tk is not homologous to the target DNA, it is lost from homologous recombinants. Thus, cells that have undergone homologous recombination are uniquely resistant to both G418 and ganciclovir, and survive in a mixture of the two antibiotics. The presence of the disrupted gene has to be confirmed by Southern blotting or by PCR using primers in the neor gene and in cellular DNA lying outside the region used in the targeting construct. By using two different resistance genes, one can disrupt the two cellular copies of a gene, making a deletion mutant (not shown).
Fig. A.45 Gene knockout in embryonic stem cells enables mutant mice to be produced. Specific genes can be inactivated by homologous recombination in cultures of embryonic stem cells (ES cells). Homologous recombination is performed as described in Fig. A.44. In this example, the gene encoding β2-microglobulin in ES cells is disrupted by homologous recombination with a targeting construct. Only a single copy of the gene needs to be disrupted. ES cells in which homologous recombination has taken place are injected into mouse blastocysts. If the mutant ES cells give rise to germ cells in the resulting chimeric mice (striped in the figure), the mutant gene can be transferred to their offspring. By breeding the mutant gene to homozygosity, offspring can be tested to determine if a mutant phenotype is generated. In this case, the homozygous mutant mice lack MHC class I molecules on their cells, because MHC class I molecules have to pair with β2-microglobulin for surface expression. The β2-microglobulin-deficient mice can then be bred with mice transgenic for subtler mutants of the deleted gene, allowing the effect of such mutants to be tested in vivo.
studied. In addition, the parts of the gene that are essential for its function can be identified by determining whether function can be restored by introducing different mutated copies of the gene back into the genome by transgenesis. The manipulation of the mouse genome by gene knockout and transgenesis has revolutionized our understanding of the role of individual genes in lymphocyte development and function.
Because the most commonly used ES cells are derived from a poorly characterized strain of mice known as strain 129, the analysis of the function of a gene knockout often requires extensive back-crossing to another strain. One can track the presence of the mutant copy of the gene by the presence of the neor gene. After sufficient back-crossing, the mice are intercrossed to produce mutants on a stable genetic background.