CAJ | 학술논문

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,800滋g/mL G-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.
위료향전기인우제공과표체 A-FABP 적공핵세포,본연구종 NCBI 중사조우지방형지방산결합단백(Albert, A-FABP)기인전장 cDNA 서렬,통과간병밀마자적방법설계특이성인물.A-FABP 기인통과공사합성,여 pEGFP-C1재체련접형성중조질립 pEGFP-C1-A-FABP.분리획득3주진천우태인성섬유세포(battle embryonic fibroblast, BEF),분별용 DMEM 화 D/F12진행배양,수궤선택일주세포우화외원기인전염조건.용우화적조건전염저3주세포,800자g/mL G-418진행사선,도취단극륭,획득은정전염 A-FABP 적세포주.PCR 화 RT-PCR 검측세포 A-FABP 기인적표체.결과현시,D/F12비 DMEM 능촉진세포적증식(p<0.05);세포재고당 DMEM 적전염효솔현저고우 D/F12(p<0.05);2주태인성섬유세포(1웅1자)획득5주은정표체적세포,기중4주래원우자성세포.결과증명세포배양기、성별화세포계영향우 A-FABP 전기인효솔.
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.