A replacement vector used to introduce subtle mutations into mouse genes

A replacement vector convenient for introducing subtle mutations into various mouse genes has been developed using, a model system, the mouse transthyretin-encoding gene (ttr) and mouse embryonal carcinoma F9 cells. The vector consists of part of ttr carrying a subtle mutation in its second exon, and a cassette of the neomycin-resistance (neo)- and herpes simplex virus thymidine kinase (HSV-tk)-encoding genes flanked with a 3-kb duplication of mostly the second intron of ttr. In the first step ('replacement'), part of the endogenous ttr was replaced by vector DNA via homologous recombination, and two such clones, #33 and #77, were isolated from 185 G418-resistant clones by allele-specific PCR. In the second step ('excision'), gancyclovir-resistant colonies were screened, and 7 and 84% of those isolated from clones #33 and #77, respectively, were demonstrated to carry the subtle mutation in ttr, without the cassette of selection markers. In five independently isolated random integrants of the same vector DNA, the cassette of selection markers was excised efficiently by recombination within the duplication.