The Studies Of Qinchuan Cattle MYOSTATIN Gene Sequence Analysis And Knockout

Myostatin ( GDF-8) gene is a negative regulator of skeletal muscle growth and development factor. The so-called double-muscled phenotype in Belgian Blue, Piedmontese, and other cattle breeds is due to deletion or mutation at the myostatin gene locus. Since the "double muscle" animal has the character of meat ratio and lean meat percentage with high, fast growing, more tender meat, etc., it makes the meat livestock and poultry industry to greater economic benefits. In order to improve the meat production performance of Qinchuan cattle, in this investigation, we compared the nucleotide composition and polymorphism of Qinchuan and Red Angus cattle breeds by sequencing. Based on this, we analysised the effect of first intron and promoter of myostatin gene on gene expression. Meanwhile, in order to get enough donor cells for RNA interference and gene targeting, we isolated fetal bovine muscle satellite cells by different methods, then purified and characterized. On the othere hands, we deleted part of nucleotides of the myostatin gene exon III in Qinchuan skeletal muscle satellite cells and fibroblaslts by gene targeting methods. Also, we used the transgenic positive cells as SCNT donor cells, then obtained the blastocyst and characterized.In this experiment, we analyzed the myostatin gene polymorphism of 16 Qinchuan and 4 Angus cattle by PCR amplification, positive and neative sequencing methods. Meanwhile, we compared the myostatin sequence with other cattle breed that reported by GenBank. The results showed that myostatin gene is highly conserved in these two cattle breeds, the homology is more than 95%. There are 1 to 4 nucleotide polymophisms in 3 exons, of which only 3 mutations on exon I caused encoded protein changed, the others are synonymous mutations. We found in 3 Qinchuan cattle that there are 16 bp consecutive nucleotides insertion mutation in after 705 nucleotides of first intron.In this study, we cloned and analyzed the sequence of the bovine myostatin gene promoter and first intron from Qinchuan and Red Angus cattle by PCR. Then constructed eukaryotic expression vectors encoding the EGFP vector by replacing the CMV promoter with the bovine myostatin promoter using PCR method, thereby obtaining an expression vector coding EGFP report gene with first intron (identified as pEGFP-MSTNPro-intron1 and pEGFP-MSTNPro). After transfected C2C12 cells 48 h later, fluorescent indices (FIs), which indicate the expression rate and intensity of gene EGFP expression, were analyzed by flow cytometry (FCM). Results showed that Qinchuan sequence homology of promoter was 99% with Red Angus, that Qinchuan first intron sequence homology was 99.51% with Red Angus and that first intron homologies of Qinchuan and Red Angus were 99.08 and 99.02% respectively, with Accession No.AF320998 in GenBank. Expression of the EGFP gene did not differ significantly between preparations using the Qinchuan versus Red Anguspromoter(P>0.05). There was no significant difference(P>0.05) in gene expression between normal first intron and 16 bp insertion first intron (+16 bp) preparations. Preparations with a construct that included the first intron had higher EGFP gene expression in C2C12 cells than those whose construct lacked the first intron(P<0.05 or P<0.01). Bovine skeletal muscle satellite cells were prepared by collagenase I, collagenase I combine togather with trypsin digestion and pronae digestion three methods. And cells were purified by using differential adhesion and Percoll density gradient centrifugation. The cells were characterized by RT-PCR and immunohistochemistry staining using Desmin antibody. The results showed that bovine skeletal muscle satellite cells were successfully harvested by three methods, but the cell number and cell viability was different. The cell number were 0.0017±0.00023, 0.5430±0.1357 and 8.1800±0.7950 separately, the cell viability were 92.67±3.79, 89.33±2.52 and 62.67±3.06 separately.We cloned fragments of Qinchuan myostatin gene as the gene targeting arm (long arm and short arm) by PCR. And deleted exon III in the short arm which included the missing 11 bp nucleotides of Belgian Blue cattle. Then constructed the gene targeting vector by replacing the two multiple cloning sites of PA2T (univesal gene targeting vector), of which containing the deletion of part of base of exon III. Then Qinchuan bovine skeletal muscle cells and fibroblasts were transfected by electroporation and treated by positive and neative screening. Extracted genome from the positive clones and characterized by PCR, Southern Bloting. The results showed that there were 2 clones that have been successfully knocked out one allele of myostatin gene. Meanwhile, the expression of myostatin protein decreased significantly of the 2 positive clones of skeletal muscle satllite cells by Western Blot.The cell cycle of 2 positive fibroblasts was analysised by flow cytometry, the results showed that the proliferation index (PrI) were 23.12% and 20.96% separately which was lower than control fibroblasts (29.80%). Both of the two clone can support the development of somatic nuclear transfer. No significant difference was found in cleavage and blastocyst development rate between this two transgenic positive cells, the cleavage rates were 61.17 and 66.36 separately, the blastocyst rates were 18.45 and 21.82 separately. But the blastocyst rate is lower in nontransfected fibroblast control group (P<0.05), furthermore, the apoptosis rate of cloned embryos inner cell derived from transgenic positive donor cells was significantly higher than the control fibroblasts group (11.66±1.84 vs 8.32±1.60,P<0.05).