Fig. 1. Targeting strategy of astrotactin locus. (A) Wild-type astrotactin locus (WTL): black rectangles represent exons. The black lines on either side represent the 5' and 3' external probes used to detect homologous recombination by Southern blot. Astrotactin targeting vector (TV): a 168 bp exon containing the translation initiation site (ATG) was designed to be replaced with a pgk1-neo cassette. pgk1-neo and HSV1-tk were used as positive and negative selection markers, respectively. (B) Targeted locus after homologous recombination (ML): BglII digests liberate 8.0 kb and 6.5 kb fragments from the wild-type and targeted alleles, respectively. PstI digests liberate 9.5 kb and 7.8 kb fragments from the wild-type and targeted alleles. SpeI digests give 10.0 kb for wild-type allele and 8.4 kb for the mutant allele. (C) Western blot analysis indicates that this mutant produces no astrotactin protein in astrotactin null animals.
