牛胎儿皮肤成纤维细胞的分离培养及转染Ipr1基因

Culture of Bovine Fetal Skin Fibroblasts in vitro and Ipr1 Gene Transfection

为了为核移植提供供体细胞,本研究构建了真核表达载体PEGFP-SRA-Ipr1,并研究了牛胎儿皮肤成纤维细胞的体外分离培养,进行了Ipr1基因转染。克隆Ipr1基因和SR-A启动子,并构建了巨噬细胞特异性真核表达载体。用组织块贴壁法分离培养牛胎儿皮肤成纤维细胞,在体外经传代、纯化后,用电穿孔法将真核表达载体PEGFP-SRA-Ipr1转染至经体外纯化的第4～10代牛胎儿成纤维细胞,24h后观察荧光表达,48h后加入600μg.mL-1 G418,筛选1周,300μg.mL-1持续筛选,然后挑选单克隆,继续扩大培养。对稳定转染的牛胎儿成纤维细胞进行PCR检测和核型分析。结果,转染24h后有绿色荧光蛋白表达,并经G418筛选获得稳定转染PEG-FP-SRA-Ipr1的牛胎儿成纤维细胞株,经PCR检测在大约1 500bp处有目的片段,经流式细胞仪分析转染细胞染色体倍型未发生变化,仍是二倍体。说明目的基因已经成功整合,同时保持了遗传稳定性。可以作为核移植进行转基因克隆牛研究。

In order to provide the donor cells for nuclear transfer,in this study,the eukaryotic expression vector PEGFP-SRA-Ipr1 was constructed,the bovine fetal fibroblast cells were isolated in vitro and the cells with PEGFP-SRA-Ipr1 were transfected.Ipr1 gene and SR-A promoter were successfully cloned and then used to construct the macrophage-specific eutherin expression vector.Bovine fetal fibroblast cells from ear were cultured in vitro by tissue explantation,passaged and purified.Then the eutherin expression vector PEGFP-SRA-Ipr1 was transfected into the cells at the passages of 4th to 10th by using electroporation.The fluorescence was observed after 24 h.600 μg·mL-1 G418 was added into the culture medium after 48 h,which lasted 1 week.After that,the concentration of G418 decreased to 300 μg·mL-1 for screening.Then the monoclones were picked and cultured for amplification.The positive cells were determined via PCR and flow cytometry.GFP was observed 24 h after transfection.The bovine fetal fibroblast cells permanently carrying PEGFP-SRA-Ipr1 were obtained after the screening of G418.The interested fragment was confirmed to be correct by PCR and the positive cells had the normal karyotype,suggesting that the interested gene was successfully integrated into the genome and it failed to disrupt the stability of the genome.In conclusion,the obtained cells could be utilized as the donor cells for research of transgenic cloned cattle by nuclear transfer.