婆罗双树样基因4 shRNA干扰载体的构建及其对THP-1细胞的转染

摘要

目的 构建针对婆罗双树样基因4(SALL4)的特异性shRNA干扰载体,并转染THP-1细胞,以期为更深入了解SALLA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SALL4特异性siRNA和一条阴性干扰siRNA,分子克隆方法构建pGPU6/GFP/Neo/SALIAshRNA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SALL4 shRNA转染细胞的SALL4表达情况,找出干扰效果最好的SALL4 shRNA.结果 4种SALL4 shRNA均能有效转染THP-1细胞,实时定量PCR(RT-PCR)与Western blotting结果均显示针对靶点mRNA-1122的pGPU6/GFP/Neo/SALL4 shRNA-B干扰效果最佳,SALL4表达减少量最多(P<0.05).结论 成功构建SALL4 siRNA干扰载体,可选择SALL4 shRNA-B载体进一步完成对SALL4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 SALL4 in leukemia. Methods Four SALL4-specific siRNA to aim at different SALL4 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 SALL4 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 SALL4 shRNA-B would be useful to accomplish study of SALL4.