Figure 2

Muscle-specific targeting of Gdf11 gene. (A) Representation of targeting strategy. The 3 exons are shown as boxes with coding sequences shaded black and the 3'UTR open. The targeting construct is represented by a thick line which contains loxP sequences with EcoRI restriction sites inserted into the second XbaI site in exon 1 and flanking a neo gene inserted into the EcoRI site downstream of the 3'UTR. Cre-mediated recombination of the loxP sites flanking the neo gene results in a Gdf11^floxallele. Recombination of the Gdf11^floxallele generates the Gdf11^Δ2–3 allele. Oligonucleotide primers used for distinguishing alleles are labeled a, b, and c. A skeletal muscle-specific Cre expressing transgene was constructed using the MLC1 promoter/1/3 enhancer and an SV40 t antigen intron and poly adenylation signal. (B) Southern blot showing detection of Gdf11⁺, Gdf11^flox, and Gdf11^-(null, from the original knockout line) alleles. (C) Northern blot analysis of MLC-Cre transgene expression in pectoralis and quadriceps muscles but not in other tissues. (D) Detection of Gdf11 alleles in genomic DNA in Gdf11^flox/floxand Gdf11^flox/floxMLC-Cre mice by PCR (right panel). Recombination was detected in quadriceps and pectoralis muscles of Gdf11^flox/floxMLC-Cre mice but not in heart, brain, or kidney. No recombination was seen in Gdf11^flox/floxmice. Left panel shows control reactions. (E) Northern blot analysis of skeletal muscle Gdf11 expression showing a strong decrease in expression in Gdf11^flox/-MLC-Cre muscle compared to Gdf11^flox/-muscle.