|Disruption of the PGC-1α Gene in Mice|
A neomycin-based gene targeting vector was generated to delete exons 4 and 5 of the murine PGC-1α gene. The targeting event resulted in a 3′ homologous recombination with insertion of the remainder of the construct (Figure 1A). The insertion/recombination event was confirmed by Southern blotting and DNA sequencing. The insertion caused an exon 3 duplication between exons 5 and 6 that creates a coding region frameshift resulting in a premature termination at amino acid 255. Germline transmission of the mutant allele was confirmed using Southern blotting (Figure 1B) and PCR (unpublished data). The PGC-1α gene disruption resulted in an unstable transcript that could not be detected by RNA blot analysis in heart and other tissues in PGC-1α−/− mice (Figure 1C and unpublished data). Quantitative RT-PCR was utilized to further evaluate the efficacy of the gene targeting. For these studies, PCR primers were designed to amplify a region of the PGC-1α gene transcript containing the exon 5–6 border (predicted to be absent in PGC-1α−/− mice) or the exon 5–3 border (predicted to be present only in the PGC-1α−/− mice). The exon 5–6 amplicon was detected in heart and BAT of wild-type (WT) but not PGC-1α−/− mice (Figure 1D). Conversely, the exon 5–3 product was present only in PGC-1α−/− mice (Figure 1D). An exon 10–11 border amplicon (predicted to be present in both genotypes) was detected in WT and PGC-1α−/− mice, but was greatly diminished in the PGC-1α−/− mice, indicating that the mutant transcript is unstable. PGC-1α protein was not detected in whole cell (Figure 1E) or nuclear protein extracts (unpublished data) isolated from BAT of PGC-1α−/− mice under basal conditions or in response to cold exposure, a condition known to markedly induce the expression of PGC-1α in BAT. Smaller mutant PGC-1α proteins were also not detected by Western blot analysis (unpublished data). Lastly, expression of the genes encoding the other known PGC-1 family members, PGC-1β and PRC, was not significantly altered in heart of PGC-1α−/− mice (Figure 1C). Taken together, these results support the conclusion that the gene targeting event resulted in a PGC-1α null allele.