核骨架基质结合区(S/MAR)提高逆转录病毒载体介导egfp在NIH3T3细胞中的表达

为减轻逆转录病毒介导的外源基因的沉默 ,进一步提高逆转录病毒MFG载体介导的转移基因的表达 ,将人 β INF基因上游 80 0bp的核骨架基质结合区 (S MAR)分别反向和正向克隆至MFG载体 3′LTR上游 ,以egfp为报告基因观察S MAR对egfp基因表达水平以及对病毒滴度的影响 .结果显示 :反向和正向的S MAR在瞬时表达的情况都不能提高egfp的表达 ,但在稳定整合的情况下反向S MAR可明显提高egfp在NIH3T3细胞内的表达 ,而正向的S MAR作用不明显 ,另外反向S MAR可明显提高MFG逆转录病毒载体的滴度约 5倍 ;因此改造后的MFG逆转录病毒载体将能更好地用来介导外源治疗基因的表达 .同时还观察到 ,同一个载体骨架在稳定表达的情况下 ,磷酸钙介导较逆转录病毒载体介导的表达水平高 .提示逆转录病毒的生活史可能参与其介导的外源基因的沉默

Scaffold Matrix Attachment Region (S/MAR) Enhances the Retroviral Vector Mediated Expression of egfp in NIH3T3 Cells

In order to relieve the retroviral vector mediated gene silencing and further enhance the expression of MFG retroviral vector mediated transgene, the 800 bp scaffold matrix attachment region (S/MAR) from upstream of human β INF gene to the upstream of 3′ LTR of MFG vector in reverse and positive orientation were cloned, respectively. And egfp gene was used as a reporter to study the effect of S/MAR on gene expression and virus production. The result showed that S/MAR could not augment the expression of egfp either in positive or reverse orientation in transient phase, while in stabilized integration S/MAR in reverse orientation enhanced the expression of egfp significantly. S/MAR was found for the first time to be capable of improving the titer of retroviral vector MFG by about 5 fold. The modified retroviral vector MFG containing a 800bp S/MAR might be used to express the therapeutic gene in a better way. In addition, there is a higher level expression in calcium phosphate mediated plasmids transfection than retroviral vector mediated infection in stabilized state, though the vector having the same structure. This result suggests that the life cycle of retrovirus maybe involved in the retroviral vector mediated transgene silencing.