The ability to disrupt genes by homologous recombination in murine embryonic stem (ES) cells and transmission of the disrupted gene through the germ line of mice after reimplantation of the stem cells into early mouse embryos has opened a new dimension in mammalian genetics. This technique is useful for the generation of animal models for human diseases on which therapeutic approaches can be tested. This chapter describes the procedures commonly employed to generate and analyze mice and ES cell lines in which the genes for endocytic cell surface receptors have been destroyed. The chapter focuses on the use of replacement type vectors that cannot be lost from the insertion site by homologous intragenic rearrangements. Replacement vectors can be constructed to allow convenient identification of homologous recombination events by polymerase chain reaction (PCR) analysis of neoR clones. The lower frequency of homologous integration of replacement vectors can be effectively compensated for by the introduction of a negative selection step that preferentially eliminates clones that arise from nonhomologous integration of the targeting vector.
ASJC Scopus subject areas
- Cell Biology