中文摘要：

目的了解牛卵丘细胞的生长特征及构建的基因载体对卵丘细胞的转染情况和转染后阳性细胞的扩增效率。方法用透明质酸酶消化牛卵丘/卵母细胞复合体（COCs）获得牛卵丘细胞,了解牛卵丘细胞的生长特点,并用分子大小分别为7178 bp和31 085 bp两种带有增强绿色荧光蛋白（EGFP）和新霉素抗性基因（Neor）的质粒载体pMSCV-EGFP-Neor和pGC1-collagen-EGFP-Neor分别转染靶细胞。结果用脂质体法转染对数生长期的细胞,均获得了绿色荧光阳性细胞,但小质粒的转染效率在72 h时是大质粒的6倍（14%vs.2.3%）。在800μg/mLG418筛选浓度下,14 d后分别获得了7个和3个较明显的单克隆细胞群,对它们进行挑选、扩大,都获得了较纯的单克隆细胞系。最后根据EGFP和collagen的已知基因序列对转染pGC1-collagen-EGFP-Neor质粒的阳性细胞进行三个不同区域DNA片段的扩增,结果表明,扩增的基因片段大小正确,数目完整。结论对卵丘细胞进行基因转染,分子大小等于或小于31 kb的基因可以有效转入,但小分子载体比大分子载体具有更高的转染效率。经过筛选都可以获得纯的转基因细胞系。

英文摘要：

Objective To understand the growth characteristics of bovine cumulus cells and the transfection efficiency of cumulus cells,based on the gene vector pGC1-collagen-EGFP-Neor that containing human collagen type I cDNA gene and mammary gland specific promoter,constructed in our laboratory.Methods Bovine cumulus cells were obtained by digestion of bovine cumulus/oocyte complexes（COCs） using hyaluronidase.Gene vector pMSCV-EGFP-Neor and pGC1-collagen-EGFP-Neor was transfected into cumulus cells with cationic liposomes.The molecular size of pMSCV-EGFP-Neor and pGC1-collagen-EGFP-Neor are 7178 bp and 31085 bp,respectively,both of them contained enhanced green fluorescent protein gene（EGFP） and neomycin resistance gene（Neor）.Results Transfection of the two plasmids generated green fluorescence positive cells.But the transfection efficiency of small molecular plasmids was 6 times（14% vs.2.3%） higher than the large molecular plasmid at 72 h of transfection.Screening used 800 μg/mL G418 at day 14,seven and three distinct positive monoclonal cell populations were obtained,respectively.The cells were selected and expanded,and pure positive cell lines were successfully obtained.Finally,the positive cell lines of transfection of pGC1-collagen-EGFP-Neor were identified by DNA amplification.The results showed that amplified gene fragments were of correct size and number.Conclusions The gene vector can be effectively transfected into cumulus cells using cationic liposomes,if molecular size of the gene is equal to or less than 31 kb.The small molecule vector has a higher transfection efficiency than the big one.However,after proper screening,both of them can be used to obtain pure transgenic positive cell lines.