ZFNS Mediated Gene Targeting On Bovine MSTN Locus

Zinc finger nuclease is a gene targeting technology that emerged recently. It can recognize specific DNA site in genome, and introduce double strand broken (DSB) to genomic DNA. The damaged DNA could be repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). When the DNA was repaired through NHEJ, nucleotides insertion or deletion may occur, and the mutations happen. While the DNA repaired through HR, it will not cause mutation, however, when the exogenous DNA fragment with homologous arms existed, the gene targeting will happen at the DSB site.Firstly, bovine fetal fibroblasts, muscle-derived mesenchymal cells and adipose-derived mesenchymal cells were dissociated and cultured from bovine fetal tissues. These cell types can be cultured and passaged in complete medium containing high glucose DMEM and10%fetal bovine serum. After the differentiation, the myotubes and adipocytes can be derived from muscle mesenchymal cells, and the adipose-derived mesenchymal cells can differentiate into adipocytes, in which the oil drops can be stained by Oil Red O. The shrinkage of the myotubes can be observed when the muscle mesenchymal cells underwent muscle differentiation. These mesenchymal cells were stained by vimentin antibody, and the results show that vimentin was expressed in these cells, they have the properties of mesenchymal cells. Furthermore, the expression of MYOG, MYOD, and MYF5can be detected in muscle mesenchymal cells. And the cells derived from bovine fetal ear have the typical characteristics of fibroblast. At last, the fibroblast and muscle mesenchymal cells were used as the main candidate for gene editing experiments.Two pairs of ZFNs (bZFNs-Pl and bZFNs-P2) aimed to the second exon of bovine myostatin (MSTN) were designed and transfected into the bovine fetal fibroblasts and muscle mesenchymal cells by lipofection. Eighteen cell lines with mutations at targeting sites were obtained after screening by polymerase chain reaction and sequencing, which include14cell lines with bZFN-P1site mutation,3cell lines with bZFNs-P2site mutation and one double alleles mutated cell line at bZFNs-P2site. The rates of gene knockout were6.05%and3.84%, respectively. The cell line KO-241was chosen to be the donor for somatic cell nuclear transfer after it confirmed has a normal karyotype. Twenty-four reconstructed blastocyst stage embryos were obtained. The trophoblast stem cell like cells were derived from these embryos after removed zona pellucida and cultured in2i medium supplied with bovine fibroblast feeder. The trophoblast stem like cells were growth in flat and dome structure. They have the same genotype at the mutation site as the donor cells.To test the gene targeting efficiency of ZFN in bovine and increase the content of unsaturated fatty acid in the skeleton muscle, two targeting donor plasmids aimed to the bZFN-P2site, named pflrkt-1and pGlrkt-1, were constructed. These two donor vectors have800-900bp homologous arms at both ends of hFat-1and EGFP. Because of the lipofection is insufficient to deliver three or more plasmids into cells simultaneously, the electroporation was employed. The highest transfection ratio60%was obtained at the condition of200V,5ms,2pulses. Using these parameters, the donor plasmid and the ZFNs plasmids were transfected into bovine fetal fibroblasts. After the G418and cross junction PCR screening, two targeted cell lines were obtained, named34f6and34GF8. The targeting ratios were10%and7%. The green fluorescence can be detected under microscope in cell line34GF8, and the expression of hFat-1also can be detected using real-time PCR in34f6, which indicating these foreign genes can express at MSTN locus.In this study, the gene mutation was introduced to bovine myostatin by ZFNs, total18mutation cell lines obtained, which include one double alleles mutation cell line. Using the donor plasmids with short homologous arms, exogenous gene EGFP and hFat-1can integrated into the MSTN locus by ZFNs, and the gene expression can be detected. The results indicated that ZFN technology can work efficiently in bovine somatic cells and may play an important role in the production of transgenic animals, which will reduce material and time costs and improve the bio-security of animal transgenic technology.