Li Hong-Mei, Yu Yue-Cheng, Song Tian-Bao, Xin Xiao-Yan, Zhang Ming, Wei Xiao-Li, Xu Jing-Hong
Department of Obstetrics and Gynaecology, Affiliated Hospital of Yanan University, Yan'an 716000, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Dec;23(12):1106-9.
To construct the TRAIL gene eukaryotic expression vector modulated by the human telomerase reverse transcription gene core promoter and to evaluate the expression of the TRAIL gene in ovarian cancer cell line SKOV3 in vitro.
The amplified TRAIL gene fragment was subsequently cloned into hTERT promoter-pIRES2-EGFP vector and CMVpromoter-pIRES2-EGFP vector. The hTERT promoter-pIRES2-EGFP-TRAIL and CMVpromoter-pIRES2-EGFP-TRAIL eukaryotic expression vectors were obtained, respectively. The plasmids were transfected into human ovarian carcinoma SKOV3 cell line by lipofeclin mediation and the positive clones were screened by G418. The expression of TRAIL gene was examined by RT-PCR, Western blot, immunocytochemistry and flow cytometry.
All the constructed vectors were verified by enzyme digestion. The TRAIL gene of transfected cells was increased significantly (P<0.01).
An eukaryotic expression vector of the TRAIL gene modulated by the human telomerase reverse transcription gene core promoter has been constructed successfully and its steady expression in human ovarian cancer cell line SKOV3, which will be beneficial to further research into its role in regulating the biological behavior of ovarian cancer cells.
构建人端粒酶逆转录基因核心启动子调控的TRAIL基因真核表达载体，并评估其在卵巢癌细胞系SKOV3中的体外表达。
将扩增的TRAIL基因片段分别克隆到hTERT启动子-pIRES2-EGFP载体和CMV启动子-pIRES2-EGFP载体中，分别获得hTERT启动子-pIRES2-EGFP-TRAIL和CMV启动子-pIRES2-EGFP-TRAIL真核表达载体。通过脂质体介导将质粒转染到人卵巢癌SKOV3细胞系，并用G418筛选阳性克隆。通过RT-PCR、Western印迹、免疫细胞化学和流式细胞术检测TRAIL基因的表达。
所有构建的载体均经酶切验证。转染细胞的TRAIL基因明显增加（P<0.01）。
成功构建了人端粒酶逆转录基因核心启动子调控的TRAIL基因真核表达载体，其在人卵巢癌细胞系SKOV3中稳定表达，这将有助于进一步研究其在调节卵巢癌细胞生物学行为中的作用。
