Rat Anti-Mouse PAX7 monoclonal antibody, clone 3G23I5

MicroRNAs are endogenous, small non-coding RNAs that can play critical gene-regulatory roles during skeletal muscle development and are highly conserved. miR-487b-3p is expressed in muscle, and the detailed mechanism by which it regulates myoblast proliferation and differentiation has not been explored. Here, we found that miR-487b-3p expression was significantly higher in goat muscle tissues than in other tissues and was higher in fetal goat muscle tissues than in mature goat tissues, suggesting that miR-487b-3p has an important effect on skeletal muscle myogenesis. Functional studies showed that miR-487b-3p overexpression significantly suppressed C2C12 myoblast proliferation and differentiation, which was accompanied by the down-regulation of functional genes related to proliferation (MyoD, Pax7 and PCNA) and differentiation (Myf5, MyoG and Mef2c), whereas the inhibition of miR-487b-3p accelerated C2C12 myoblast proliferation and differentiation and was accompanied by the up-regulation of functional genes. Using Target-Scan and David, we found that miR-487b-3p targeted the 3'-UTR of IRS1, an essential regulator in the PI3K/Akt and MAPK/Erk pathways. We then confirmed the targeting of IRS1 by miR-487b-3p using dual-luciferase assays, RT-qPCR and western blotting. Furthermore, IRS1 silencing markedly inhibited proliferation and differentiation in cultured C2C12 myoblasts, confirming the important role of IRS1 in myogenesis. These results reveal an IRS1 -mediated regulatory link between miR-487b-3p and the PI3K/Akt and MAPK/Erk pathways during skeletal muscle myogenesis.

CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9. Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%-47% and 24%-89% of Pax7(+) MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9. Cre to Pax7-ZsGreen-Ai14 mice in which Pax7(+) MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9. Cas9 and AAV9. gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin- null NSG. mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin(+) cells (p = 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy.