Research Of Myostatin Gene Target And Transcriptional Regulation In Pig

Myostatin, also named the growth and differentiation factor8(GDF8), is amember of the tumor growth factor-β super-family, Myostatin is a secreted proteinthat acts as a negative regulator of skeletal muscle mass. Natural Myostatin genemutations occur in cattle showing the double-muscling phenotype. Genetic deletion ofMyostatin in mice leads to excessive muscle growth by hyperplasia and hypertrophyof muscle fiber. Pig breeding and pork production in China rank number one in theworld. The pig is also the most important source of meat supply in China. Pigs can beused as an ideal animal model of studying human disease due to their more similarityto human in the physiology, anantomy, and genetics. The production of the pigs withdeletion of Myostatin gene has implications in both agriculture for improvingbreeding, increasing the carcass lean meat percentage of the livestock andbiomedicine as a laboratory animal model for human diseases such as musculardystrophy.In this study, we first constructed pig Myostatin homologous recombinationvector pFlexible-DT-MSTN-SA-LA. To inactivate Myostatin gene, the third exon ofpig Myostatin gene was mutated. The pig fetal fibroblasts were transfected with thevector and selected for positive cell clones with puromycin drug. After nucleartransfer and embryo transfer,5live Myostatin knockout pigs were achieved,identified by PCR and sequence. Further analysis showed that one of the alleles ofMyostatin gene was mutated. The difference of Myostatin gene expression levelbetween the Myostatin gene knockout pigs and wild type pigs was not significant(P=0.1734>0.05; P=0.2956>0.05), confirmed by real-time PCR and Western blot.Further study on transcription regulation mode of Myostatin gene found thatpromoter structure in mice and men were relatively close. The structure of Myostatingene promoter region of cattle and sheep were most similar. The structure ofMyostatin promoter of pigs was closer to cattle and sheep than to man and rat. FoxO1、SMAD2and MyoD transcription factor binging sites on the promoter regionwere found in all these five species, while cdxA transcription factor binding sites wasoberserveed only in cattle and sheep. The expression level of FoxO1、SMAD2andMyoD transcription factors was not significantly different(P=0.7369>0.05;P=0.9165>0.05;P=0.5939>0.05) between Myostatin gene knockoutpigs and in wild type pigs, confirmed by real-time fluorescent quantitative PCR. Thismay explain why there was no phenotypes difference between Myostatin geneknockout pigs and wild type pigs. The lack of signicant muscle mass increasing inMyostatin gene knockout pigs may be because single gene inactivation was not ableto lead to feedback inhibition of transcription factor expression. The left the copy ofMyostatin gene in the other chromosome could compensate expression of mayostatinnormal level.The results here may lay foundation to further study on the mechanism ofregulation and evolution of Myostatin gene in future.