适用于shRNA表达研究的克隆载体构建

目的:建立一种适用于shRNA表达研究的克隆载体。方法:通过PCR扩增一段含有自杀基因ccdB和卡那霉素抗性基因的DNA序列,将其双酶切后克隆至shRNA表达载体pSilencer3.1-H1neo中,得到重组体pSilencer3.1-ccdB。运用此重组体构建荧光素酶的shRNA表达载体并设立对照进行非重组背景的结果比较。结果:成功建立了pSilencer3.1-ccdB克隆载体。应用此载体构建荧光素酶的shRNA的表达载体,结果显示完全消除了非重组背景。结论:建立了适用于shRNA表达研究的克隆载体,为进一步研究siRNA的基因功能奠定了基础。

Construction of a cloning vector appropriate for shRNA expression study

Objective:To establish a novel vector with low non-recombinant background and high success rate of cloning and thus suitable for shRNA expression study.Methods:The DNA sequence containing ccdB suicide gene and kanamycin resistance gene was amplified by using PCR and cloned into the shRNA expression plasmid pSilencer3.1-H1 neo that was previously treated with appropriate restriction endonucleases.The power of the resulting vector pSilencer3.1-ccdB was evaluated by cloning the luciferase shRNA expression sequence.As control,the original parent vector pSilencer3.1-H1 was also employed to clone the sequence.Results:The pSilencer3.1-ccdB vector dramatically increased the success rate of cloning by removing the non-recombinant background as compared with the control vector.Conclusion:This study provides a useful tool that will prove its efficacy in the studies of gene function using RNA interference techniques.