人睾丸特异表达基因TDRG1shRNA表达载体的构建及其表达

目的:构建人睾丸基因TDRG1 的shRNA 表达质粒, 并研究其在NTE-2 细胞中的表达。方法:设计合成有短发夹结构靶位TDRG1 基因的寡核苷酸, 经退火成双链, 克隆至载体pGPU6/GFP/Neo 中, 构建TDRG1shRNA 表达载体, 得到重组质粒TDRG1-shRNA486, TDRG1-shRNA738, TDRG1-shRNA921 和一个阴性对照, 使用Lipofectamine^TM 2000 转染shRNA 至NTE-2 细胞, 应用RT-PCR 法检测转染后NTE-2 细胞TDRG1 mRNA 的表达水平。结果:经测序证实, 插入的DNA 片段的序列与设计序列完全一致。同时筛选到转染TDRG1-shRNA486 的NTE-2 细胞TDRG1 基因的mRNA 表达水平明显抑制。结论:成功构建了人类睾丸基因TDRG1 的shRNA 表达载体, TDRG1-shRNA 在NTE-2 细胞内成功表达。

Construction of TDRG1 shRNA expression vector and interfering effect of TDRG1 shRNA expression vector on NTERA-2 cells

Objective: To construct short hairpin RNA interfering expression vector of TDRG1,and detect thespecific interfering effect of TDRG1-shRNA expression vector on NTERA-2 cells.
Methods: Oligos for short hairpin RNA targefing for TDRG1 were designed and connected tothe expression vector pGPU6/GFP/Neo to construct the TDRG1 shRNA expression vector.The recombinant plasmid TDRG1-shRNA486, TDRG1-shRNA738, TDRG1-shRNA921 andlipofectamine^TM 2000 were used to generate and transfect shRNA into NTERA-2 cells. Expressionof TDRG1 mRNA was assayed by RT-PCR.
Results: TDRG1-shRNA expression vector was successfully constructed. TDRG1-shRNA486 wasmore effective in the suppression of TDRG1 with significant reduction of TDRG1 mRNA.
Conclusion: TDRG1-shRNA can interfere the expression of TDRG1 in NTERA-2 cells.