Key Laboratory of Animal Reproductive Endocrinology & Embryo Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi 712100, China.
Res Vet Sci. 2012 Oct;93(2):763-9. doi: 10.1016/j.rvsc.2011.10.022. Epub 2011 Nov 23.
Myostatin is an important negative regulator of muscle growth and development. Natural mutations of the myostatin gene cause a double muscling phenotype in beef cattle, pigs and sheep. Therefore, it is feasible to produce a high growth domestic breed by generating a transgenic animal with a mutation, deletion or knockout of the myostatin gene. Our objective was to introduce a subtle mutation of G to A 281-bp upstream of the 3' untranslated region (3'UTR) end of the myostatin gene in Poll Dorset fetal myoblast cells in vitro. Fetal myoblast cells were isolated from fetuses at day 50 of gestation from Poll Dorset sheep and transfected with linear gene-targeting vector pMSTN-A using electroporation. We obtained seven gene-targeted cell colonies with homologous recombination, which were positive as confirmed by PCR, Southern blot. The Western blot analysis result demonstrated that the myostatin protein expression in positive colonies is lower than that of negative ones. These results strongly suggest that we successfully mutated the myostatin gene of Poll Dorset ovine fetal myoblast cells and the mutation can effectively downregulate the myostatin protein expression.
肌肉生长抑制素是肌肉生长和发育的重要负调控因子。肌肉生长抑制素基因的自然突变导致肉牛、猪和羊出现双肌表型。因此，通过产生具有肌肉生长抑制素基因突变、缺失或敲除的转基因动物，生产生长速度快的本土品种是可行的。我们的目的是在体外将 Poll Dorset 胎儿成肌细胞中的肌肉生长抑制基因 3'UTR 末端上游 281bp 的 G 突变为 A。从 Poll Dorset 绵羊妊娠第 50 天的胎儿中分离胎儿成肌细胞，并用电穿孔法将线性基因靶向载体 pMSTN-A 转染到细胞中。我们获得了 7 个具有同源重组的基因靶向细胞集落，通过 PCR 和 Southern blot 证实为阳性。Western blot 分析结果表明，阳性集落中的肌肉生长抑制素蛋白表达低于阴性集落。这些结果强烈表明，我们成功地突变了 Poll Dorset 绵羊胎儿成肌细胞中的肌肉生长抑制素基因，该突变可有效下调肌肉生长抑制素蛋白的表达。
