REST/NRSF基因shRNA慢病毒载体的构建与鉴定

Construction and identification of lentiviral vector encoding shRNA against REST/NRSF

背景:神经元限制性沉默因子(REST/NRSF)能负向调控神经元及胰岛细胞分化相关基因的表达。目的:构建并筛选能高效沉默大鼠REST/NRSF基因的shRNA慢病毒载体。方法:针对REST/NRSF基因设计4组特异性siRNA靶点,合成靶序列的寡核苷酸序列,退火形成双链DNA,与经HpaⅠ和XhoⅠ双酶切后的pFU-GW-RNAi载体连接产生L-shREST/NRSF慢病毒载体,PCR筛选阳性克隆,测序鉴定。包装产生慢病毒颗粒,随后将其感染大鼠骨髓间充质干细胞,采用Real-timePCR方法检测靶基因在mRNA水平的沉默效率。结果与结论:PCR和测序证实,构建出了REST/NRSFshRNA的慢病毒载体L-shREST/NRSF,并能稳定转染大鼠间充质干细胞,感染效率达100%。4组shRNA序列均有基因沉默效果,并以第3组shRN序列效果最为明显。结果表明,该慢病毒表达载体能够在细胞水平有效沉默靶基因。

BACKGROUND： In recent years, many studies showed that respressor element1（RE-1） 1-silencing transcription factor /neurons restrictive silence factor （REST/NRSF） could negative control neurons and islet cell differentiation related gene expression. OBJECTIVE： To construct a lentiviral vector expressing shREST/NRSF. METHODS： Four groups of shRNA sequences specifically targeting the REST/NRSF gene were designed and synthesized and cloned into the pFU-GW-RNAi vector. The obtained lentiviral vector containing shREST/NRSF was confirmed by PCR and sequencing. 293T cells were cotransfected with lentiviral vector L-shREST/NRSF, pHelper1.0 and pHelper2.0.The titer of virus was tested according to the expression level of GFP. And preliminary observations on the situation of transfected rat bone marrow mesenchymal stem cells. Real time PCR was employed to assess the gene silencing efficacy of these recombinants. RESULTS AND CONCLUSION： PCR and DNA sequencing demonstrated that the constructed lentivirus vector L-shNRSE/RE-1 produced REST/NRSF shRNA. And it could be stably transfected to rat bone marrow mesenchymal stem cells, there was the infection efficiency of almost 100%. All of these four shRNAs could achieve gene knock down effect, and 3# shRNA had the most significant gene silence effect among them. The lentivirus vector of shREST/NRSF is constructed successfully.