Nesprin基因RNAi慢病毒载体的构建

背景：Nesprin蛋白缺失将影响细胞骨架组织和动态平衡,引起细胞骨架刚性丧失或导致细胞过早成熟老化,其对间充质干细胞的作用如何？目的：构建Nesprin蛋白siRNA慢病毒载体,并转染骨髓间充质干细胞.方法：针对Nesprin靶基因序列设计并合成4对miRNA oligo,将4种miRNA干扰质粒转入大鼠血管平滑肌细胞,筛选最有效干扰序列;将最佳干扰序列和pDONR221载体进行重组反应,获得含干扰序列的入门载体,再将入门载体和慢病毒表达目的载体pLenti6/V5-DEST进行重组反应,获得含干扰序列的慢病毒表达载体,转染包装细胞293T细胞,包装慢病毒,以293T细胞GFP蛋白水平测定病毒滴度.慢病毒转染大鼠骨髓间充质干细胞.结果与结论：测序证实合成的4对miRNA oligo正确,RT-PCR和western-blot筛选出最佳干扰miRNA质粒为SR-3,成功构建了Nesprin siRNA的慢病毒载体LV-siNesprin.包装慢病毒,浓缩病毒悬液的活性滴度为106 TU/mL.慢病毒成功了转染骨髓间充质干细胞细胞.

Construction of Nesprin genome RNAi lentiviral vector

BACKGROUND： Nesprin protein absence can affect the cytoskeletal tissue and homeostasis, causing the loss of cytoskeleton rigidity or leading to premature aging of cells. In this study, we investigated the effect of Nesprin protein on bone marrow mesenchymal stem cells through designing Nesprin siRNA lentiviral vector. OBJECTIVE： To construct Nesprin-siRNA lentiviral vector. METHODS： According to the target gene sequence of Nesprin, four pairs of miRNA oligo were designed and annealed into double-stranded DNA identified by sequence. siRNA interference with the four kinds of plasmids （SR-1, SR-2, SR-3, SR-4） were transformed into rat vascular smooth muscle cells to screen the most effective sequence; In order to get the sequence started with interfering carrier by RT-PCR and western-blot, we let the best interfering sequence carriers and pDONR221 to react together. Then entry vectors and lentiviral vectors to express the purpose of pLenti6/V5-DEST response was to restructure the sequence containing interference lentiviral expression vector. Lentiviral vector containing interfering sequence was co-transfected 293T cells to package lentivirus. The virus titer was determined by the expression level of GFP protein in 293T cells. Lentivirus was used to transfect rat bone marrow mesenchymal stem cells. RESULTS AND CONCLUSION： Sequencing confirmed the successful construction of Nesprin siRNA lentiviral vector LV-siNesprin. The best interference with miRNA plasmid selected by RT-PCR and western-blot was SR-3. The virus titer for lentiviral packaging and concentrating suspension of the activity was 106 TU / ml. The expression of Nesprin protein decreased in transfected cells.By certain conditions, Nesprin-siRNA lentiviral vector can be successfully constructed.