用于筛选shRNA的新型双萤光素酶报告基因载体的构建及应用

目的:构建一个新型双萤光素酶报告基因载体用于准确高效地筛选有效抑制靶基因表达的shRNA。方法:采用PCR、定向克隆、基因重组等分子生物学方法,将双萤光素酶报告基因系统中需要的报告基因载体、shRNA真核表达载体和内参载体三个功能,整合于一个新型的双萤光素酶报告基因载体pFLuc-C-TK-RLuc-shRNA之中。应用该载体对靶向人PD1基因以及Furin基因的shRNA进行干涉效果比较。结果:应用p FLuc-C-TK-RLuc-shRNA载体进行单质粒转染方法与传统的三质粒转染方法均提示shRNA#1对靶基因PD1的抑制效果最优;但是使用新型双萤光素酶报告基因载体检测结果的标准差数值显著低于传统方法。以pFLuc-C-TK-RLuc-shRNA单质粒转染方法得到的各组样品结果的均一性显著提高,能够准确地反映出shRNA#3较shRNA#2具有更强的Furin基因抑制作用;而传统方法未能有效判断二者的差异。结论:新型双萤光素酶报告基因载体简化了操作步骤,降低了传统三质粒共转染方法所引起的检测结果大幅波动,进一步增强了双萤光素酶报告基因系统的信噪比,提高了筛选抑制靶基因表达shRNA的准确性。

Construction and Application of a Novel Dual-Luciferase Reporter Gene Vector for shRNA Screening

ABSTRACT Objective: To construct a novel dual-luciferase reporter gene vector for accurate and efficient shRNA screening. Methods: Using standard molecular biological methods of polymerase chain reaction, directional cloning and gene recombination, a novel dual-lu- ciferase reporter gene vector namely pFLuc-C-TK-RLuc-shRNA was constructed. This recombinant vector integrated functions of the re- porter gene vector, the eukaryotic expression vector of shRNA and the reference vector. Target gene (PD1 or Furin) and candidate shRNAs were respectively directional cloned into the multiple cloning site one and two of the new-established vector. The interference effects of shRNAs on target gene were compared with standard dual luciferase reporter gene assay. Results: Compared to the traditional method of cotransfected with three plasmids, the new established shRNA screening method using pFLuc-C-TK-RLuc-shRNA only needs to transfect a single plasmid. In the experiment which target gene was PD1, similar results were obtained by the two competitive methods; yet, the standard deviations of each shRNA group detected by pFLuc-C-TK-RLuc-shRNA were significantly lower than that of traditional method. The results obtained by the new method have superior uniformity, which can accurately reflect the difference of the inhibition ef- ficiency of shRNA#2 and shRNA#3 targeting Furin gene. However, this trend has not been detected by traditional method of cotransfected with three plasmids. Conclusion: By alleviating the fluctuation of results obtained by traditional method, the novel reporter gene vector pFLuc-C-TK-RLuc-shRNA enhances the dual-luciferase system signal-to-noise ratio, and improves the accuracy of shRNA screening.