ABSTRACT
Gene targeting, in which DNA constructs are introduced into specific sites in the genome of ES cells, and subsequently animals, has become a powerful tool for elucidating gene function by generating animals with loss of function - so-called 'knockouts'. Until recently, homologous recombination was the only method for gene targeting in mammals. But this method is fastidious and costly and, with the exception of heterozygous 'knockouts', is not readily adaptable to analysis of the effects of partial down-regulation of gene expression. Conventional plasmid-based targeting requires the generation of complex targeting constructs, the selection of homologous recombination events in ES cells, the production of chimeric mice after microinjection of modified ES cells into blastocysts, and the establishment of pure breeding lines, before obtaining homozygous mutant mice. An alternative tool for ablating gene function in eukaryotes discovered in the past few years utilizes double-stranded RNAs (dsRNAs). This new method, called 'RNA interference', or RNAi, could well compete in many instances with homologous recombination to ablate gene function in mammalian cells.
