人Ⅰ型胶原基因脂质体转染牛耳细胞克隆筛选方法

The Research of Screening Method for HumanⅠType Collagen Gene was Transfected into the Ear Fibroblast of Holstein Cow with the Liposome

构建连接有人Ⅰ型胶原基因的质粒（PGC1-collagen）,利用脂质体介导的方法转染荷斯坦奶牛耳成纤维细胞,优化一系列转染条件以提高转染效率,再利用G418和GFP荧光的表达来筛选稳定转染的细胞,最后以未转染的成纤维细胞作为阴性对照组,PGC1-collagen作为阳性对照组对转染的细胞进行PCR验证。结果显示：在6孔培养板中细胞融合度达到50%~60%左右介导1~3μg重组质粒转染24 h,转染效率可达25%~30%,筛选单克隆细胞在荧光显微镜观察检测到绿色荧光蛋白的表达。在使用800 U的G418筛选效果最好,维持浓度300~600 U,获得25个稳定表达的阳性细胞系,从中选取6个克隆进行PCR验证。成功利用脂质体转染方法将人Ⅰ型胶原基因成功转染到荷斯坦奶牛耳成纤维细胞,PCR验证结果正确。

In the article first PGCl-colleage was reconstructed with constructed expression vector and the human Ⅰ type collagen gene , and then the ear fibroblast of Holstein cow was transfected in the liposome-mediated method, optimized a series of transfection conditions to enhance the transfection efficiency ,the stable monoclone cell line expressed the green fluorescent protein which was observed with G418 and fluorescence microscope, the normal untransfected fibroblast was the control, before transfection the authenticated archae-plasmid was the positive control, then amplified and cultivated, at last extracted the genome DNA and PCR authentication. The result indicated that the cell degrees of fusion was about 50%-60%, mediated 1-3 ug recombinant plasmid, transfeced 24 h, transfection efficiency was 25%-30% in the 6-hole culture plates, monoclone cell was screened by that detected the expression of the green fluorescent protein with fluorescence microscope. In our laboratory before the experiment, did the cell G418 tolerance experiment, indicated that 800 U G418 caused drug-resistance and screening effect was the best, maintained concentration 300 U-600 U. 25 green fluorescent protein positive clone was selected from the ear fibroblast of Holstein cow, selected 6 clone to do PCR experiment： authenticated the screened clone , extracted the genome DNA , PCR result accord with the expectant band, indicated that human Ⅰ type collagen gene was transfected into the ear fibroblast of Holstein cow in the liposome-mediated method, screened the positive clone that expressed the gene stably. PCR authentication established some foundation for the transgene clone animal.