摘要
目的筛选四环素诱导细胞周期素B1（CyclinB1）可控表达的293单克隆细胞株（tetracycline-regulated expression 293 cells，T-REx^TM-293）。方法从人胎肝cDNA文库中PCR带有酶切位点的CyclinB1基因全长，将其酶切后插入到pcDNA4／TO／myc-HisB载体中。然后用测序正确的质粒转染T-RExTM-293细胞，加杀稻瘟菌素（Blasticidin）和腐草霉素（Zeocirt）双药物筛选两周后，传96孔板，等单个细胞扩增出单克隆。然后用Western印迹和流式细胞仪检测CyclinB1的诱导表达情况。结果构建好的pcDNA4／TO／myc-HisB—CyclinB1载体，经鉴定序列正确。筛选出的单克隆细胞株，在未加四环素时没有外源性的CyclinB1表达，在加入四环素后，3h就有表达，随时间的增加，CyclinB1表达量也增加，48h最多。结论筛选出的T-REx^TM-293单克隆细胞株能可控表达CyclinB1。
Objective In this study we sought to establish a monoclone cell line expressing Cy- clin B1 induced by tetracycline. Methods The full sequence of Cyclin B1 was obtained from fetal liver cDNA library by PCR. After overnight digestion with BamH1 and X-ball, the digested prod- ucts of Cyclin B1 and empty vector pcDNA4/TO/myc-HisB were ligated overnight with T4 DNA lig- ase. The resultant pcDNA4/TO/myc-HisB-Cyclin B1 was sequenced and transfected into T-RExTM- 293 cells. The transfected cells were selected in DMEM culture medium containing the selective markers of blasticidin and zeocin for two weeks, and then seeded into 96 well plates for colony growth. After induction of cyclin B1 for different time by tetracycline, ceils were harvested, and Cy- clin B1 expression was tested by western blot and flow cytometry. Results The pcDNA4/TO/myc- HisB-Cyclin B1 vector was successfully constructed with the right insert of the sequence. The selected monoclonal cell lines expressed Cyclin Bl-myc-his at detectable level as early as 3 hours and high level 48 hours after tetracycline added. Conclusions The monoclonal T-RExTM-293 cell line can express Cyclin B1 under conditional expression.
出处
《医学分子生物学杂志》
CAS
CSCD
2009年第4期307-311,共5页
Journal of Medical Molecular Biology
基金
国家重点基础研究发展规划项目（973计划）子课题（No.2004CB518705,2009CB521802）,国家自然科学基金（No.30872472,30800569）,新世纪人才支持计划（No.NCET-04-0699）
