Laboratório de Sinalização e Plasticidade Celular, Departamento de Biofísica, Universidade Federal do Rio Grande do Sul-UFRGS, 91501-970 Porto Alegre, RS, Brazil.
Plasmid. 2012 Nov;68(3):179-85. doi: 10.1016/j.plasmid.2012.06.001. Epub 2012 Jun 20.
Gene transfer based on lentiviral vectors allow the integration of exogenous genes into the genome of a target cell, turning these vectors into one of the most used methods for stable transgene expression in mammalian cells, in vitro and in vivo. Currently, there are no lentivectors that allow the cloning of different genes to be regulated by different promoters. Also, there are none that permit the analysis of the expression through an IRES (internal ribosome entry site)-- reporter gene system. In this work, we have generated a series of lentivectors containing: (1) a malleable structure to allow the cloning of different target genes in a multicloning site (mcs); (2) unique site to exchange promoters, and (3) IRES followed by one of two reporter genes: eGFP or DsRed. The series of the produced vectors were named pLR (for lentivirus and RSV promoter) and were fairly efficient with a strong fluorescence of the reporter genes in direct transfection and viral transduction experiments. This being said, the pLR series have been found to be powerful biotechnological tools for stable gene transfer and expression.
基于慢病毒载体的基因转移允许外源基因整合到靶细胞的基因组中,使这些载体成为哺乳动物细胞中体外和体内稳定转基因表达最常用的方法之一。目前,没有能够克隆不同基因并由不同启动子调控的慢病毒载体。也没有能够通过 IRES(内部核糖体进入位点)-报告基因系统分析表达的载体。在这项工作中,我们生成了一系列包含以下内容的慢病毒载体:(1)一个可塑结构,允许在多克隆位点(mcs)中克隆不同的靶基因;(2)一个独特的位点,用于交换启动子;(3)IRES 后面跟着一个报告基因:eGFP 或 DsRed。所产生的载体系列被命名为 pLR(代表慢病毒和 RSV 启动子),在直接转染和病毒转导实验中,报告基因具有很强的荧光,效率相当高。可以说,pLR 系列已被发现是用于稳定基因转移和表达的强大生物技术工具。