17 Generation of myostatin gene knockout boars by somatic cell nuclear transfer

Skeletal muscle is the most economically valuable tissue part in meat-producing animals, and enhancing muscle growth in these species may increase the efficiency of meat production. Skeletal muscle mass is negatively regulated by myostatin (MSTN), and nonfunctional mutations of this MSTN gene in various animal species have led to dramatic hypermuscularity. A porcine fetal fibroblast cell line with biallelic MSTN mutations (MSTN −/−), consisting of a 2-bp deletion in one allele and a 4-bp deletion in the other allele, was used as a donor to generate cloned pigs via somatic cell nuclear transfer. Cloned embryos were transferred into seven surrogates and 38 live piglets were delivered. Using these MSTN null piglets, the detailed morphological, gene and protein expression analyses were performed not only in biceps femoris, semitendinosus, and diaphragm of MSTN null piglets but also in heart, liver, spleen, lung, kidney, and tongue. The results showed that MSTN −/− boars have visually clear hypermuscular characteristics, which was supported by the increased carcass dressing percentage and loin eye size and decreased backfat thickness. The fibre cross-sectional areas of the semitendinosus and semimembranosus muscles were much larger in MSTN −/− piglets than in wild-type (WT) piglets (98.0±8.2 vs. 62.7±4.7, 105.7±11.6 vs. 72.3±8.4mm2, respectively; P<0.05). MSTN −/− pigs showed a higher proportion of fast-type fibres (Type-II fibre%, 95±1.8 vs. 86±2.4; P<0.05) and lower succinate dehydrogenase activity in muscles than WT pigs. The mRNA expression of myosin heavy-chain IIB in both two muscles was higher in MSTN −/− pigs compared with WT pigs (P<0.05). In organs, the heart and liver were lighter in MSTN −/− piglets than in WT piglets (8.1±0.3 vs. 10.3±0.4, 26.9±1.8 vs. 34.2±1.2g kg−1, respectively; P<0.05), whereas the tongue was heavier in MSTN −/− piglets than in WT piglets and myofibres of the tongue were significantly larger in the former piglets than in the latter piglets (P<0.01). Messenger RNA expression of MSTN in all organs was significantly lower in MSTN −/− piglets than in WT piglets (P<0.01) and follistatin in the heart and liver was significantly higher in MSTN −/− piglets than in WT piglets (P<0.05). Protein expression of MSTN in the heart, kidneys, and tongue was significantly lower in MSTN −/− piglets than in WT piglets (P<0.01). In conclusion, MSTN −/− pigs showed a phenotype of supermuscular ratio is associated with increased muscle fibres and smaller internal organs and has a complicated gene expression patterns.