Multipurpose modular lentiviral vectors for RNA interference and transgene expression
Multipurpose modular lentiviral vectors for RNA interference and transgene expression
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DOI：
10.1007/s11033-009-9840-8
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发表时间：
2010-07-01
影响因子：
2.8
通讯作者：
Fuerst, Dieter O.
中科院分区：
文献类型：
--
作者：
Kesireddy, Venu;van der Ven, Peter F. M.;Fuerst, Dieter O.
我们已经创建了一个多用途的模块化慢病毒载体系统，用于表达转基因和基于miRNA30的短发夹(ShRNAmir)用于RNAi。所得到的载体系统的核心pLVmir允许一个简单的两步克隆过程，以在Pol II启动子的控制下以结构性和条件性方式表达shRNAmir。改进的克隆方法包括用于从公共可获得的文库(开放生物系统)转移基于shRNAmir的RNAi克隆的无PCR方法。Pol II启动子驱动的shRNAmir盒的增加以及Pol III启动子和沉默触发器的广泛选择为该系统提供了极大的灵活性。这里创建的几个预先存在的模块和附加模块的组合迎合了研究人员的共同需求。我们的模块化载体系统在启动子的功能性、诱导性和可逆性方面得到了验证。我们成功地应用该系统敲除了H2kb-tsA58肌肉细胞中Xirp2mRNA的表达，并确定这对一个密切相关的蛋白的表达没有虚假影响。最后，我们的一组慢病毒载体可以用来实现协同效应，同时敲除两个基因，作为救援质粒，并在生理环境中研究突变蛋白。
We have created a multipurpose modular lentiviral vector system for expressing both transgenes and miRNA 30-based short hairpins (shRNAmirs) for RNAi. The core of the resulting vector system, pLVmir, allows a simple two step cloning procedure for expressing shRNAmirs under the control of a Pol II promoter in both a constitutive and conditional manner. The adapted cloning method includes a PCR-free method for transferring shRNAmir based RNAi clones from a publicly available library (Open Biosystems). The addition of a Pol II promoter-driven shRNAmir cassette and broadening the choice of Pol III promoters and silencing triggers offers great flexibility to this system. The combination of several preexisting and additional modules created here caters to common needs of researchers. Our modular vector system was validated regarding functionality of promoters, inducibility and reversibility. We successfully applied the system to knockdown Xirp2 mRNA expression in H2kb-tsA58 muscle cells and determined that this had no spurious effect on the expression of a closely related protein. Finally, our set of lentiviral vectors may be used to achieve synergistic effects, for simultaneous knockdown of two genes, as a rescue plasmid and for studying mutant proteins in a physiological context.