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Fig. 1. Generation of mice lacking Ccm1. (A) Targeting strategy to generate the Ccm1^tm1Dmar allele. Mice heterozygous for the allele will be referred to as Ccm1^+/-, and mice homozygous for the allele will be referred to as Ccm1^-/-. Our construct was designed to replace most of the sixth and the entire seventh coding exon of mouse Ccm1 with an internal ribosomal entry site (IRES) and the E. coli lacZ gene. Although this construct successfully ablated Ccm1 gene expression, we were unable to detect any ß-galactosidase protein or enzyme activity in Ccm1^+/- or Ccm1^-/- embryos. (B) Southern blotting of KpnI-digested genomic DNA detects a 3.1 kb band from the recombinant allele of a heterozygous animal. This same probe detects a 4.3 kb band from the parent strains (C57BL/6J, labeled B6, and 129X1Sv/J, labeled 129), from the wild-type allele of a heterozygous animal and from a wild-type littermate. Long-range PCR and sequencing of the resulting product also confirmed homologous recombination and conserved sequence at the 3' and 5' ends. (C) Genotyping of mice was performed using allele specific PCR primers. The wild-type primers amplify a 466 bp product, and the mutant primers amplify a 310 bp product. (D) In situ hybridization for Ccm1 detects ubiquitous expression at E8.5 in a wild-type embryo. No transcript is detected in Ccm1^-/- embryos using this probe, which spans exons 3 through 7. Scale bars: 200 µm.

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