【作者】 王春生； 李晶； 张滕子； 宁方勇； 朴善花； 安铁洙；

【Author】 WANG Chun-sheng,AN Tie-zhu College of Life Science,Northeast Forestry University,harbin 150040,China

【机构】 东北林业大学生命科学学院；

【摘要】 为了探讨拟蜘蛛丝蛋白基因二聚体(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,本研究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,其结果,原代培养的幼鼠卵巢的颗粒细胞在培养后的第4d达到85%汇合,经继代培养1～7代,每代达到85%的汇合时间为约为3d,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浓度;与对照组不同,转染线性化的CMV-2S-pIRES2-EGFP的转染颗粒细胞后用G418浓度为500μg/mL浓度条件筛选12d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。上述结果,为进一步开展转拟蜘蛛蛋白基因小鼠颗粒细胞的核移植提供依据。更多还原

【Abstract】 In order 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 link 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 4d to congregate 90%confluence,and cells spent 3d to congregate 85% confluence in subculture.Morphology present polygon shuttle was not changed during subculture. Optimization concentration of G418 was 500μg/mL to select cells.Compare to control group,linearization CMV-2S-pIRES2-EGFP transfected cells were still active after G418 selecting 12d,but capability of proliferation lost gradually.G418 resistance cells were identified by PCR method.The result showed that the vector constructed was integrated into mouse genome.The transgenic mouse granular cell line with artificial synthesized spider dragline silk protein gene would be used in the future transgenic mouse study.更多还原