Conditional gene targeting.

Abstract

Gene targeting: The classical approach Central to an understanding of the in vivo function of genes is their analysis by mutation, that is, inactivation or modification of a gene by mutation and the study of the consequences of the mutation in the mutant organism. In mammals, before gene targeting, this approach was limited to the rare spontaneous mutations reflected in obvious phenotypes, as in the case of in- heritable diseases in the human. The targeted mutagenesis of the mouse germline was thus a fundamental breakthrough in this area of research. In its original form, gene targeting in- volves the inactivation of a given gene in the genome of em- bryonic stem (ES) cells by homologous recombination (1-3). ES cells derive from an early stage of mouse development and have retained their totipotency, thus they can participate in the generation of cell lineages of the mouse (including germ cells) if transferred into an early mouse embryo. Transfer of mutant ES cells into mouse embryos thus allows the transmission of the mutation in question into the mouse germline. In the classical experiment of Thomas and Capecchi (3) gene inactivation was achieved by replacing a predetermined gene segment with a mutant version of this segment, through homologous recombination. Since the latter is infrequent in mammalian cells, the isolation of the mutant ES cells requires stringent selection. This was achieved by placing a selectable gene (in that case the neomycin resistance gene) into the tar- geted locus in a manner that allows its expression (and hence cellular selection) while inactivating the target gene. Refining classical gene targeting by the Cre-loxP recombination system A limitation of classical gene targeting comes from the pres- ence of a selection marker gene in the targeted locus. Since this gene must be active in order to allow ES cell selection, it must always be considered that through its expression it might affect the mutant phenotype in an unpredictable way. A par-