Fig. 1. Generation and external phenotypes of Cv2 knockout mice. (A) Construction of the targeting vector. In the mutant allele, the first methionine of Cv2 was replaced in-frame with that of tau-lacZ. The locations of PCR primers used for selection of recombinant ES cells (blue arrows) and genotyping (white arrowheads), and probes for Southern blot analysis (black and orange boxes) are indicated. Bg, BglII; Sc, SacI; Xb, XbaI. (B,C) Genomic PCR and Southern blot analyses for the homologous recombinant ES cell line. (B) Recombination at the 3' region of the Cv2 genome (resulting in a 3 kb PCR fragment) was observed. (C) Correct integration was further examined by Southern blot using the 5' probe (wild-type, heterozygous and homozygous, from left to right lanes). (D) PCR genotyping of mutant mice. The wild-type allele band (564 bp) and the mutant allele band (412 bp) were amplified by PCR. (E) RT-PCR analysis of Cv2 expression in E12.5 mutant mice. (F,G) External appearances of P0 neonates (F) and E15.5 embryos (G). The control mice are on the left side and the null mutant mice are on the right side. At a low penetrance, exencephaly was also observed (arrows) at E15.5 (about 7% of embryos; n=86).