2001•Zhongguo shengwu huaxue yu fenzi shengwu xuebaoRequires access
Construction and cell line selection of target-expressing retroviral expression vector containing cDNA of TNFR75
Xiaowen Sun, Li Liu, Ying Shi, Lizhong Liu, Baoshu Xie, Ye Wang, Yan Ling Yang, Leng Aijun, Ying Cao
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Abstract
The cDNA for TNFR75 and the promotor of kinase domain receptor (KDRp) were recombinated with retroviral expression vector pLXSN-D_(299) without promotor. The recombinated plasmid was transfected into packaging cell PA317 using lipofectin method. The transfectants were selected by G418 (600 mg/L) for 14 days and 15 stable cell clones grew. Viral supernatants of the clones were checked by NIH 3T3 cells to detect the titer of the virus. The results showed that the highest viral titer among 15 clones was 2X10~(5) CFU/ml. Total RNA of the clones with the highest viral titer were extracted. RT-PCR analysis showed that cDNA amplified had the same length with that of TNFR75. It indicated that the cell line which could produce virus containing TNFR75 was constructed.