Generation and analysis of <i>Sdhd</i>-deficient mice.

<p><b>A.</b> Schematic diagram of the strategy used to target the mouse <i>Sdhd</i> locus. The structure of the endogenous murine <i>Sdhd</i> gene (wildtype allele -wt) is shown in the middle with the targeting vector above and the disrupted allele below. Genomic DNA is represented by narrow horizontal lines with exons (shaded boxes), orientation of transcription (arrows), translation initiation and stop codons indicated; Genomic sequences flanking the betaGeo selection-reporter cassette (open box) in the targeting vector are represented by broad lines. Primers for genotyping (small arrows) and RT-PCR (arrowheads) are indicated below and above their target sequences, respectively. The dumbbells indicate the location of Southern blot probes used. <b>B.</b> Long-range PCR analysis of <i>Sdhd</i> gene targeting. The 7.1 kb normal allele amplified by primers LR-F and LR-R1 is present in the wildtype (lane 1) and heterozygous <i>Sdhd</i> knockout mouse (lane 3), whereas the 8.1 kb betaGeo targeted <i>Sdhd</i> allele amplified by primers LR-F and LR-R2 is present in the heterozygous <i>Sdhd</i> knockout mouse (lane 4) and not in the wildtype (lane 2) M = 10kb ladder marker. <b>C.</b> RT-PCR analysis of targeted gene expression. The C57BL/6J wt <i>Sdhd</i> allele is represented by 603bp and 391bp bands, and a 129P2/Ola wt allele, the 994bp band. Lanes 1 & 2; F1 pups from a C57BL/6J wt×129P2/Ola <i>Sdhd</i>+/− cross show only a single C57BL/6J wildtype <i>Sdhd</i> allele. Lanes 3, 4 & 5 = controls. Lane 3: C57BL/6J wt mouse. Lane 4: 129P2/Ola wt mouse. Lane 5: 129P2/Ola wt×C57BL/6J wt F1 mouse. The absence of a 129P2/Ola wildtype allele in the F1 offspring of the 129P2/Ola <i>Sdhd</i>+/−×C57BL/6Jwt cross demonstrates that they carry the <i>Sdhd</i>/betaGeo targeted allele, indicating correct targeting of the 129P2/Ola <i>Sdhd</i> locus. M = 100bp marker. <b>D.</b> Routine <i>Sdhd</i> genotyping of pups. M = marker, lanes 1 & 3; wt mice, primers WT-F & WT-R. Lanes 2 & 4; heterozygote mice, primers WT-F & bG-R.