Abstract：Myostatin gene(MSTN) is a negative skeleton muscle development control gene, and mutation of this gene will induce the over development of skeleton muscles, which is extremely valuable for the breeding of the meat animals. In this study, two pairs of zinc finger nucleases (ZFN) aimed at bovine (Bos taurus) MSTN second exon were used to introduce the mutation in bovine MSTN gene to achieve the purpose of knocking out this gene. Firstly, the transfection condition by two plasmids to bovine fibroblasts via lipofectamine was optimized using different combination of DNA and transfection reagent, and the highest transfection efficiency was 19.25%. Secondly, the bovine fibroblasts were transfected with two pair of ZFNs, respectively; single cells were picked to 96-well plate by mouth-pipetting. After PCR screening, eighteen MSTN mutated cell lines were obtained, which included one double allele mutated cell line. The mutation efficiencies of these two pairs of ZFNs were 6.05% and 3.84%, respectively. Finally, one of the mutated cell lines was used as the donor cells for somatic cell nucleus transfer (SCNT), and 24 reconstructed blastocysts were obtained. The trophoblast stem-like cells were derived after the zona pellucida removed and cultured in 2i medium. They had the same mutation at MSTN locus with the donor cells according to the PCR and sequencing analysis. In conclusion, ZFNs were very efficient to introduce the mutation to bovine MSTN gene in fibroblasts, and they had the ability to generate double allele mutated cells in one experiment, and the mutated cells were capable to generate the reconstructed embryos for transfer after SCNT. This study provides basic data for the breeding of the beef cattle by MSTN gene mutation.
