正向和反向SV40 poly(A)信号序列对上游基因表达的影响

Influence of two kinds of SV40 poly (A) signals on upstream gene expression

目的研究正向和反向SV40poly(A)信号在3种常用细胞株中对上游基因表达的调控作用,为基因表达研究中载体poly(A)信号的选择提供依据。方法将F-Luc与R-Luc两种荧光素酶基因插入同一质粒,构建带有双荧光素酶报告基因的载体Dual-Luc。在F-Luc基因后分别插入正向和反向互补的SV40poly(A)信号序列,得到2个带有不同SV40poly(A)信号的载体Dual-Luc2、Dual-Luc3。将其分别转染常用的3类细胞株293、L-02和HeLa细胞,应用双荧光素酶标仪和反转录聚合酶链反应(RT-PCR)法测定报告基因(F-Luc)的表达量,以R-Luc基因的表达量作对照。结果成功构建携带正向和反向互补SV40poly(A)序列的双荧光素酶载体Dual-Luc2、Dual-Luc3;双荧光素酶标仪检测表明,在293细胞中,Dual-Luc2、Dual-Luc3的F-Luc/R-Luc比值分别为3.25±0.43、3.03±0.14,差异无统计学意义(P>0.05);在L-02细胞中,Dual-Luc2、Dual-Luc3的F-Luc/R-Luc比值分别为6.16±0.39、3.83±0.39,差异有统计学意义(P<0.05);在HeLa细胞中,Dual-Luc2、Dual-Luc3的F-Luc/R-Luc比值分别为1.21±0.10、0.66±0.02,差异有统计学意义(P<0.05)。RT-PCR检测与荧光素酶检测结果一致。结论不同细胞中poly(A)信号序列对上游基因的调控作用存在差异;poly(A)信号对上游基因的调控作用主要发生于转录水平。

Objective To investigate the regulatory effects of two kinds of SV40 poly（A） signals on the upstream gene expression in three cell lines,so as to provide theoretical evidence for selection of poly（A） of vectors.Methods A dual luciferase reporter vector Dual-Luc was constructed,and two SV40 poly（A） signal sequences were inserted in the downstream of the R-Luc gene separately.Then the two diverse dual luciferase reporter gene vectors Dual-Luc2 and Dual-Luc3 were transfected into 293,L-02,and HeLa cells.The relative quantities of the target gene （F-Luc） to control gene （R-Luc） were measured by Dual-GloTM Luciferase Assay System and Reverse Transcription-Polymerase Chain Reaction （RT-PCR）.Results The two Dual-Luciferase vectors （Dual-Luc2 and Dual-Luc3） were successfully constructed.Dual-GloTM Luciferase Assay showed that the mean F-Luc/R-Luc values were 3.25±0.43 and 3.03±0.14 in Dual-Luc2 and Dual-Luc3 transfected 293 cells（P0.05）,6.16±0.39 and 3.83±0.39 in L-02 cells（P0.05）,and 1.21±0.10 and 0.66±0.02 in HeLa cells（P0.05）,respectively.The results of RT-PCR were consistent with those of luciferase assay.Conclusion The two kinds of SV40 poly（A） signals have different regulatory effects on the upstream gene expression in different cell lines.SV40 poly（A） signals regulate the upstream gene expression at the transcriptional stage.