Fig. 1. Targeting of the mouse Brdt locus. (A) Targeting construct used to generate Brdt mutant mice. The top line depicts the genomic region of the Brdt gene, along with the PCR primers and probes used for genotyping targeted embryonic stem (ES) cells and subsequent progeny; the middle line depicts the targeting construct with a Neo and TK cassette; the bottom line indicates the organization of the resulting recombined allele. (B) Northern hybridization revealed that the mutant allele is still transcribed but produces an mRNA that is smaller in size (3.4 kb). (C) Western blot showing that the shorter mRNA is translated in frame to produce a protein that is recognized by our -CT antibody. (D) Splicing of the mutant Brdt mRNA and the predicted corresponding protein product. Reverse transcription (RT)-PCR of the mutant testicular mRNA was used to define the exact nature of the mRNA and the putative truncated protein. The mRNA results from splicing from exon 1 to exon 5; there is an in-frame ATG at the beginning of exon 5. The original translation start codon (ATG) is located in exon 3, which has been deleted in the mutant allele. The allele that produces this altered mRNA is referred to as Brdt^BD1.