摘要
为了向转基因牛提供过表达A-FABP的供核细胞,本研究从NCBI中查找牛脂肪型脂肪酸结合蛋白(Albert,A-FABP)基因全长cDNA序列,通过简并密码子的方法设计特异性引物。A-FABP基因通过公司合成,与pEGFP-C1载体连接形成重组质粒pEGFP-C1-A-FABP。分离获得3株秦川牛胎儿成纤维细胞(battle embryonic fibroblast,BEF),分别用DMEM和D/F12进行培养,随机选择一株细胞优化外源基因转染条件。用优化的条件转染这3株细胞,800μg/mLG-418进行筛选,挑取单克隆,获得稳定转染A-FABP的细胞株。PCR和RT-PCR检测细胞A-FABP基因的表达。结果显示,D/F12比DMEM能促进细胞的增殖(p<0.05);细胞在高糖DMEM的转染效率显著高于D/F12(p<0.05);2株胎儿成纤维细胞(1雄1雌)获得5株稳定表达的细胞,其中4株来源于雌性细胞。结果证明细胞培养基、性别和细胞系影响牛A-FABP转基因效率。
In order to provide donate cells with over expression of （A-FABP） for transgene battle, full-length cDNA sequence of A-FABP was examined in NCBI GenBank, to design specific primers by degenerate codon approach. The A-FABP gene synthesized by commercial company was aligned to pEGFP-C1 vector to make a recombinant plasmidPEGFP-C1-A-FABP. Threestains ofembryonic fibroblast were isolated and cultured in the media of EMEM and D/F12, respectively. One of them was randomly chosen and applied to optimized the condition of transgene. The optimized condition was applied to transfect the three cell stains. Consequently, transfected cell was screened by G-418 with 800 滋g/mL, and the formed colony with GFP was picked up. The cell expressed A-FABP gene was identified with PCR and RT-PCR. Results showed that sequencing of reconstructed plasmid of A-FABP was ac-cording to the expectation. The proliferation of cells in D/F12 medium could be promoted than that of DMEM （p〈0.05）.Transfection rate of cells in DMEM was higher than that of D/F12 （p〈0.05）. From two （one was male and the other was female） of three cell strains, 5 cell stains with stable expression of A-FABP was obtained, 4 of which were derived from female embryonic fibroblast. The results demonstrated that the rate of transfection of exogenous gene could be impact by cell medium, gender and cell strains.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2012年第6期544-548,共5页
Genomics and Applied Biology
基金
国家自然科技基金(青年基金)(30900155)
自然科学基金(81070496)
陕西省教育厅基金(09JK785)
西北大学生命科学学院重点实验室基金(2009)共同资助
