拟蜘蛛拖丝蛋白基因在小鼠颗粒细胞中的整合及其鉴定

Integration and identification of mouse granular cells with the spider dragline silk protein gene

为了探讨拟蜘蛛拖丝蛋白基因二聚体（2S）在小鼠颗粒细胞基因组中整合和建立转拟蜘蛛拖丝蛋白基因的小鼠颗粒细胞系的可能性,试验将在前期工作中人工合成的拟蜘蛛拖丝蛋白基因2S连接到具有CMV启动子、IRES序列和GFP报告基因的表达载体,并转染小鼠颗粒细胞。结果表明：原代培养的幼鼠卵巢的颗粒细胞在培养后的第4天达到85%汇合,继代培养1-7代时每代达到85%的汇合时间约为3 d,细胞的形态与原代培养细胞相似,呈现多角形或梭形;筛选阳性细胞的最佳G418浓度为500μg/mL;将线性化的CMV-2S-pIRES2-EGFP转染颗粒细胞,再用500μg/mLG418筛选12 d后仍有存活细胞,但是续培养细胞的增殖能力逐渐丧失;经PCR鉴定,阳性细胞扩增出目的条带。

To discuss the feasibility of establishment of mouse granular cell line with the transgene of spider dragline silk protein gene,in this experiment artificially synthesized spider dragline silk protein gene was linked to the pIRES2-GFP vector with CMV promoter,IRES sequence and GFP report gene,and granular cell was transfected with the recombination plasmid.The results showed that natal mouse primary granular cell spent 4 d to congregate 90% confluence,and cells spent 3 d to congregate 85% confluence in subculture.The cell morphology was similar to that of the original cultured cells,showing polygonal or spindle-shaped during subculture.Optimization concentration of G418 was 500 μg/mL to select cells.Compare to control group,linearization CMV-2S-pIRES2-EGFP transfected cells were still active after G418 selecting 12 d,but capability of proliferation lost gradually.G418 resistance cells were identified by PCR method.The result indicated that the vector constructed was integrated into mouse genome.It concludes that the transgenic mouse granular cell line with artificial synthesized spider dragline silk protein gene can be used in the future study of transgenic mouse.