FIG. 1.

Generation and analysis of KifC2 mutants in ES cells. (A) Strategy for generating KifC2 knockout mice. One 4.0-kb DNA fragment between BamHI and EcoRI was replaced with a β-Gal-PGK-neo-pA cassette. This cassette contains a promoterless β-Gal, which should use the KifC2 endogenous promoter to drive expression, and a complete PGK-driven neo gene. The N-terminal 30 amino acid residues of KifC2 are fused to the third amino acid of β-Gal. The targeting vector was linearized with NheI and introduced into RI ES cells as described in Materials and Methods. (B) Southern analysis of ES cells. ES cell genomic DNA was cut with EcoRI and probed with a 1.5-kb SmaI-EcoRI DNA fragment shown in Fig. 1A. The wild-type and targeted KifC2 alleles were detected as 4.0- and 5.5-kb bands, respectively.