摘要
目的构建针对婆罗双树样基因4（SALIA）的特异性shRNA干扰载体，并转染THP-1细胞，以期为更深入了解SALIA对白血病的作用，为后续研究提供实验工具。方法设计4条针对不同靶点的SALIA特异性siRNA和-条阴性干扰siRNA，分子克隆方法构建pGPU6／GFP／Neo／SALIA-shRNA干扰载体，转染THP-1细胞，比较未转染细胞、阴性干扰转染细胞和4条SALIAshRNA转染细胞的SALIA表达情况，找出干扰效果最好的SALIAshRNA。结果4种SALIAshRNA均能有效转染THP-1细胞，实时定量PCR（RT—PCR）与Westernblotting结果均显示针对靶点mRNA-1122的pGPU6／GFP／Neo／SALIAshRNA—B干扰效果最佳，SALIA表达减少量最多（P〈O．05）。结论成功构建SALIAsiRNA干扰载体，可选择SALL4shRNA—B载体进-步完成对SALIA基因功能的研究工作。
Objective To construct an efficient SALL4 shRNA vector and transfect it into THP-1 cells for investigating the effect of SALIA in leukemia. Methods Four SALIA-specific siRNA to aim at different SALIA mRNA target sites and a negative control siRNA were designed, and pGPU6/GFP/Neo/SALL4 shRNA vectors were constructed. THP-1 cells were transfected and the expression of SALIA in shRNA detected, and blank control and negative control were also designed. Results The results of real time quantitive PCR and Western blotting both exhibited that the interference effect of pGPU6/GFP/Neo/SALL4 shRNA-B vector was optimal targeting to mRNA-1122 target site and down-regulated the expression of SALL4 more significant（P 〈0.05）. Conclusion Successfully construction of SALL4 siRNA vector by choosing SALIA shRNA-B would be useful to accomplish study of SALL4.
出处
《白血病．淋巴瘤》
CAS
2010年第1期12-15,共4页
Journal of Leukemia & Lymphoma
