Figure 1. Generation and analysis of Sdhd-deficient mice.

A. Schematic diagram of the strategy used to target the mouse Sdhd locus. The structure of the endogenous murine Sdhd 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. Long-range PCR analysis of Sdhd 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 Sdhd knockout mouse (lane 3), whereas the 8.1 kb betaGeo targeted Sdhd allele amplified by primers LR-F and LR-R2 is present in the heterozygous Sdhd knockout mouse (lane 4) and not in the wildtype (lane 2) M = 10kb ladder marker. C. RT-PCR analysis of targeted gene expression. The C57BL/6J wt Sdhd 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 Sdhd+/− cross show only a single C57BL/6J wildtype Sdhd 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 Sdhd+/−×C57BL/6Jwt cross demonstrates that they carry the Sdhd/betaGeo targeted allele, indicating correct targeting of the 129P2/Ola Sdhd locus. M = 100bp marker. D. Routine Sdhd 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.