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首页> 外文期刊>BMC Biotechnology >Modified gateway system for double shRNA expression and Cre/lox based gene expression
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Modified gateway system for double shRNA expression and Cre/lox based gene expression

机译：用于双shRNA表达和基于Cre / lox的基因表达的改良网关系统

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Background The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations. Results Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA) and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein- EGFP , yellow fluorescent protein-YFP and red fluorescent protein-dsRed2). Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines. Conclusion Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.
机译：背景技术对基因功能研究的需求日益增长，为开发基因操纵的多功能工具奠定了基础。结果为了为基因功能的高通量分析提供工具，我们开发了基于Gateway Technology的改良的短发夹RNA（shRNA）和基因表达系统。该系统包含一系列进入和目的载体，可将shRNA或cDNA轻松转移到具有多种选择或标记基因（即嘌呤霉素，潮霉素，绿色荧光蛋白-EGFP，黄色荧光蛋白-YFP和红色荧光蛋白）的慢病毒表达系统中蛋白-dsRed2）。我们的shRNA进入载体pENTR.hU6.hH1包含两个串联的人shRNA表达启动子H1和U6，能够同时共表达两个shRNA序列。构建基因过表达的进入载体pENTR.CMV.ON，使其包含CMV启动子，该启动子具有多个克隆位点，两侧为loxP位点，可用于随后的Cre / lox重组。 shRNA和cDNA表达载体都还包含与我们的目标表达载体中的attR位点重组所必需的atLL位点。作为原理证明，我们通过测试多种细胞系中若干cDNA和shRNA序列的表达来证明该系统的功能和效率。结论我们的系统是基于网关的载体的现有库的宝贵补充，并且可以是基因功能研究许多方面的必不可少的工具。

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