| Myostatin (Myostatin, MSTN) is an important negative regulator of skeletal muscle growth, and is a member of the transforming growth factor-(3(TGF-β) superfamily. It can inhibit the differentiation and growth of muscle. It has been reported that low-level expression of MSTN gene through genetic operations could accelerate the growth of muscle and increase the production of meat. In this study, we constructed for MSTN gene Targeting Vectors with TALENs, and optimized condition that was best for plasmid DNA transfected into cultured the Arbas Cashmere goat fetal fibroblast cells by liposomal. Then the MSTN specific transcriptional activator like effector nucleases (TALENs) targeting vectors were transfected into this cell line under optimal transfection condition. MSTN+/-and MSTN-/-cells were screened by PCR and sequencing. The MSTN-/-genotype cells were used for somatic cell nuclear transfer (SCNT) to prepare embryos and produce MSTN knockout Cashmere goats.1. Optimization of co-transfection condition for goat fetal fibroblastsLiposome co-transfection condition of Cashmere goat fetal fibroblasts was optimized by using red fluorescent protein expression vector pCMV-DsRed and green fluorescent protein expression vector pEGFP-C1. After48hours of transfection, both the red and green fluorescence could be observed in a large number of the cells under inverted fluorescence microscope. Comparison of transfection efficiency between available transfection reagents, we found it was optimal condition for liposome co-transfection method that adding250ng pCMV-DsRed plasmid,250ng pEGFP-C1plasmid and3μL Lipofectamine to every1×105fetal fibroblast cells of Cashmere goat, resulted with a highest transfection efficiency of24.5%as shown by the FACS analysis.2. Preparation of the MSTN gene knockout goat fetal fibroblast cells by TALENsIn this study, the four-unit and three-unit module were assembled together after enzyme cleavage, and finally a pair of TALENs vectors were successfully constructed by using the "unit assembly"method. TALENs targeting vector bMK (S1586-PEAS, S1586-PERR) were transfected into Arbas Cashmere goat fetal fibroblast cells using the optimized transfection condition. A single cell clone techniques was performed to established monoclonal cell lines of fetal fibroblast. Monoclonal cell lines were identified by sequencing of PCR products amplified with primers across TALENs sites, and the target point mutation monoclonal cell lines were screened out. In this study,160monoclonal cell lines were obtained from three gene targeting experiments, including20monoallelic knockout cell lines and3biallelic knockout cell lines, with a total knockout efficiency of14.38%. The chromosome number of D4, one of the biallelic knockout cell lines, was analyzed, with a normal cell rate up to90%(27/30). In this study, the D4cell line was used as the donor cells during somatic cell nuclear transfer.3. Generation of MSTN gene knockout Cashmere goatsGoat ovaries were collected from slaughtering house, and the oocytes were obtained using the cutting method and were cultured18hours in vitro. Blind suction method was used for removing the oocyte nucleus, and the MSTN-/-and WT cell lines as donor cells were immediately transferred into the enucleated oocytes. After electro-fusion and chemical activation, the cloned embryos were cultured in vitro. Electric fusion was used for the fusion of cloned embryos, with the fusion rates of74.59%and71.2%respectively. IA23187and6-DMAP were used for chemical activation. All embryos were cultured with embryo culture medium, and48h later it was observed that the cleavage rates were65.54%and65.9%, respectively. In this study,738MSTN-/-type reconstructed embryos and723WT-type reconstructed embryos were obtained by using the SCNT.363cleavage MSTN-/-type embryos were transplanted into84recipients’ oviducts at the third day since natural estrus. Two months later, we found that seven ewe recipients were pregnant by pregnancy test, with the pregnancy rate of4.76%. |
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