Fig. 2

Brinp1 targeting. a The Brinp1 targeting vector was designed with a neomycin resistance cassette after exon 3, and FRT sites positioned before and after the neo^r cassette. The 190 bp third exon of Brinp1 contains the start of the MACPF domain. LoxP sites flank exon 3 and the neo^r cassette. When crossed with a mouse line expressing Cre-recombinase, the recombination of LoxP sites resulted in the deletion of exon 3 and the neo^r cassette. b Genomic DNA isolated from the spleen was cleaved with Pst I and Bgl III and hybridised to 500 bp genomic DNA probes from the 5′ region (Pst I) and 3′ region (Bgl III) of the targeting construct. In wild-type DNA, species of 7.1 kb (Pst I) and 18.4 kb (Bgl III) were detected. These products were not present in DNA from Brinp1 ^−/− mutants, replaced with shorter species of 5.4 kb (Pst I) and 11.4 kb (Bgl III). c cDNA from the brain tissue of WT or Brinp1 ^−/− mice was tested for exon 3 deletion by RT-PCR. Primers designed to regions of exon 2 and exon 6 resulted in a PCR product size corresponding to the removal of the 190 bp exon 3 for Brinp1 ^−/− cDNA. d Sequencing of the Brinp1 ^−/− allele RT-PCR product showed the expected absence of exon 3, and that splicing fuses exons 2 and 4, resulting in a frame shift that introduces a stop codon after 24 residues. e By immunoblotting, full-length 85 kDa BRINP1 was present in lysates of mouse brains at postnatal day 12 and absent in Brinp1 ^−/− mice