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Fig. 1. Targeted inactivation of the Col4a1/2 locus. (A) The murine Col4a1/2 genes are arranged head-to-head with a common shared promoter element (P), exons (shaded squares) and relevant restriction sites (E, EcoRI; Xh, XhoI; Nh, NheI; H, HindIII) are indicated. The targeting construct containing the pgk/neomycin resistance cassette (Neo) and the mutant locus (bottom) are shown. Probes P1, P2 and the detected EcoRI fragments (arrows) are marked. (B) Genotyping of ES cells and newborns by Southern blotting. DNA was isolated, digested with EcoRI and hybridized with probe P1. The 15 kb and 9.5 kb bands are the wild-type and mutant alleles, respectively. (C) Genotyping of E10.5 embryos by PCR. The 0.7 and 0.4 kb fragments represent the mutant and wild-type alleles, respectively. Markers are indicated (kb). (D) Detection of {alpha}1(IV) and {alpha}2(IV) mRNAs in embryos at E9.5 and E10.5 by RT-PCR. Annexin A5 (Anxa5) was used as an internal control.