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Fig. 1. Targeting of cingulin alleles by homologous recombination in mouse ES cells. (A) Restriction maps of the cingulin genomic locus [wild-type (WT) allele] and the neomycin (Neo) and hygromycin (Hyg) targeting vectors. Putative exons 2-5 are represented by black boxes. A 1.98 kb KpnI-XbaI fragment beginning 775 bp upstream of the ATG translation start codon and ending 353 bp downstream of the 3' end of exon 2 was replaced by a neomycin-resistance cassette in the first targeted allele and by a hygromycin resistance cassette in the second targeted allele. In the targeting vectors, the antibiotic resistance cassettes were flanked on the 5' side by a 5.5 kb XhoI-KpnI intron fragment (left arm) and on the 3' side by a 2.4 kb XbaI-EcoRV fragment (right arm) containing intronic sequences and exons 3-5. Positions of the 5' and 3' probes, and the neomycin and hygromycin probes used for Southern-blot analyses are shown as thick lines. A, ApaI; E, EcoRI; E5, EcoRV; K, KpnI; Xb, XbaI; Xh, XhoI. (B-G) Southern-blot analysis. Restriction endonucleases used for digestions are indicated beneath each blot. Genotypes and probes are indicated above each blot. Sizes (kb) of hybridizing bands are indicated on the left.

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