Figure 1  Deletion of the PGC-1α Gene
(A) Schematic of the gene targeting strategy. A region of the murine PGC-1α gene containing exons 3–6 is shown schematically at the top. Relevant restriction endonuclease sites are also shown. The targeting construct containing a neomycin (Neo) cassette is shown below the PGC-1α gene with dashed lines indicating the regions targeted for homologous recombination. Homologous recombination between the 3′ end of the targeting vector and the PGC-1α gene is indicated by the solid lines. The targeting construct inserted into the PGC-1α gene resulting in a duplication of exon 3 and incorporation of the targeting construct DNA into the final recombinant as shown. Probes used for the Southern blot studies and relevant restriction fragments predicted by digestion of the recombinant are also shown.
(B) Southern blot analysis of embryonic stem cells (ESC) (digested with Xba1) and tail DNA (digested with Pst1) is shown. The blots were hybridized with the probes shown in Figure 1A. Results for PGC-1α+/+ (+/+), PGC-1α+/− (+/−), and PGC-1α−/− (−/−) genotypes are shown as denoted at the bottom.
(C) Northern blot analysis using RNA isolated from the hearts of the three relevant PGC-1α genotypes (as in Figure 1B) is shown using PGC-1α cDNA as a probe. In addition, PGC-1β and PRC cDNA probes were used as shown. Ethidium bromide staining of 18S ribosomal RNA is shown at the bottom.
(D) Quantitative real time RT-PCR (Sybr green) was used to detect PGC-1α transcripts using primer sets crossing different exon borders as denoted. The exon 5–3 primer set detects only the mutant transcript (−/−), whereas the exon 5–6 primer set detects only the WT transcript. The values represent arbitrary units for RNA isolated from the tissues shown for the three amplicons comparing PGC-1α+/+ and PGC-1α−/−. N.D. = not detectable. The exon 10–11 amplicon was evaluated to assess levels of mutant versus WT transcripts. The values represent arbitrary units normalized to actin control.
(E) Western blot analysis using whole-cell protein extracts prepared from BAT under basal conditions and following exposure to 4 °C for 8 h. The signal shown was obtained with polyclonal anti-PGC-1α antibody [10]. An epitope-tagged PGC-1α, overexpressed in neonatal cardiac myocytes using an adenoviral vector (Ad-PGC-1α), is shown as a positive control. The Ponceau S stain of the protein gel is shown at the bottom.