A Preliminary Study On The Knockout Of Myostatinin Buffalo Somatic Cells

Buffalo is a major livestock in the south of China. With the development of productivity, the labor of buffalo has been partially replaced by machine. At the same time, buffalo's meat and milk have attracted more and more attention, myostatin is a key gene which plays a great role in regulating the growth of muscle mass. The meat yield of animal would be improved if the myostatin gene was knocked out. In order to improve buffalo's meat performance and breed a new breed, the present study was undertaken to explore the possibility of gene targeting in buffalo somatic cells.Firstly, two pairs of primers were designed according to the sequence of myostatin of bovine which has been issued in GeneBank. Then, the long (4368bp) and short (1374bp) homologous arms were amplified from the local buffalo genome by the long fragment PCR technique. Sequencing and homologous analysis indicated that the long arm has 97% homology and the short arm has 96% homology with the bovine. These data proved that the two fragments was part of buffalo myostatin gene and were suitable for the targeting vector constructing. The homologous long arm contains the exon I, II and part of intronII fragment, while the short arm contains part of exon III of myostatin gene. By linking the two arms to ploxp, a myostatin knockout vector named as pl3K had been consturcted. The vector was identified to be identical to the design by enzyme digestion and PCR examination.Then, using the improved explant and trypsin digestion methods, buffalo fetal fibroblast cells were efficiently isolated from the fetal's skin, and had been cultured in vitro for three generation.Finally, the myostatin knock-out vector was transfected into the buffalo fetal fibroblasts by lipofectamine-mediated and electric pulse-mediated methods, and the cells resisting to G418 and GANC were selected by culture in the medium containing G418 or GANC. The parameters of transfection, the selection and culture condition of transfected cells were optimized. Lastly, these cells resisting to G418 and GANC were identified by PCR examination. In conclusions, (1) 8 μl of lipofectamine, 2μg of DNA and 8h of transfection are optimum for lipofectamine-mediated gene transfection; As for electric pulse-mediated method, (2) high transfection rate can be achieved when 45V/mm of DC intensity and 12ms of pulse duration were employed in the electric pulse-mediated methods; (3) DMEM + 20%FBS + 10μg/ml insulin + 10ng/ml bFGF + 30% conditioned medium are suitable to culture transfected cells; (4) integration of p13K into the cell genome may be in a random way.