Generation And Phenotypic Analysis Of Myostatin Gene Editing Meishan Pigs

Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there have not been reported significant muscle phenotypes in pigs associated with MSTN mutations. In addition to an important source of meat production, pig serves as a preferred animal model for human disease as well. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs without marker gene via zinc finger nucleases (ZFN) technology.Primary porcine fetal fibroblasts were established from fetus collected from a 35-day pregnant Meishan sow, then, the cells were transfected with the ZFN plasmid pairs which were specificially designed to target exon2 of MSTN gene using nucleofector. After cultured for 24h, the cells were collected and total DNA were extracted. The target fragments were obtained by PCR and used for TA cloning sequencing. Depending on the sequencing results of 1000 random bacterial colonies, the targeting efficiency of this ZFNs was determined as-4%. Subsequently, after fetal fibroblasts were transfected with the ZFNs, single-cell colonies were produced by limiting dilution in drug-free cell culture medium. We screened 1800 cell colonies using sequencing method. Of these 1800 cell colonies, 80 MSTN-mutant cell colonies were identified, including 78 heterozygous mutation (MSTN+/-) and 2 homozygous mutation (MSTN-/-) identified by DNA sequence analysis. Over 90% of these mutants were short fragment deletions or insertions (< 20bp). Fourteen MSTN-mutant cell colonies were selected as donor cells to produce MSTN-mutant piglets by using SCNT (somatic cell nuclear transfer), and then 2631 reconstructed embryos were transferred to 28 receptor sows. A total of 19 mutant piglets were born live from cell colony#105,#110 and#127. Among these mutant piglets,8 piglets from cell colony #105 and 1 piglet from cell colony#110 grew to adulthood.The MSTN+/- male cloned pigs were mated with WT pigs to generate Fl female and male MSTN+/-pigs. F1 heterozygous pigs were then bred to generate F2 MSTN-/-, MSTN+/- and MSTN+/+ pigs. The MSTN-mutant pigs developed and grew normally, and had increased muscle mass (MSTN-/-:p<0.01, MSTN+/-:p<0.05) with decreased fat accumulation compared with MSTN+/+ pigs (MSTN-/-:p<0.001, MSTN+/-:p<0.05), and the homozygote MSTN mutant (MSTN-/-) pigs had apparent DM phenotype, the lean percentage was greater in MSTN-/- pigs by 11.62% than that in MSTN+/+ pigs, and individual muscle mass increased by 100% over their wild-type controls (MSTN+/+) at eight months of age as a result of myofiber hyperplasia. Interestingly,20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.