Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases.
Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases.
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DOI：
10.1186/1472-6750-11-107
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发表时间：
2011-11-16
影响因子：
3.5
通讯作者：
Harper SQ
中科院分区：
文献类型：
--
作者：
Garwick-Coppens SE;Herman A;Harper SQ
RNA干扰(RNAi)是由小抑制性microRNAs(MiRNAs)介导的一种保守的基因沉默机制。启动子驱动的miRNA表达载体已经成为将天然或人工设计的miRNA运送到真核细胞和生物体的重要工具。这样的系统可以用来询问天然miRNAs或Messenger RNAs的正常或致病功能，或者用于治疗沉默疾病基因。与任何分子克隆程序一样，构建基于miRNA的表达结构需要时间投资和一些分子生物学技能。为了提高效率和加快构建过程，我们开发了一种利用重组酶快速构建miRNA表达载体的方法，取代了传统的剪切粘贴分子克隆技术。除了简化构建过程外，我们的克隆策略还为载体提供了更多的多功能性。在我们的系统中，miRNAs可以从U6启动子中组成性表达，也可以通过Cre重组酶诱导表达。我们还设计了一种内置机制，如果需要的话，可以用FLP重组酶摧毁载体。最后，为了进一步简化构建过程，我们开发了一个软件包，使用我们的系统自动预测和设计最佳miRNA序列。我们设计并测试了一个模块化系统来快速克隆miRNA表达框。我们的策略减少了成功生成有效构建所需的实际操作时间，并且可以在具有最少分子克隆专业知识的实验室中实施。根据最终用户的需要，这个通用的系统提供了允许组成性或诱导性miRNA表达的选项。因此，它对基本或翻译应用程序具有实用价值。
RNA interference (RNAi) is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs). Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes. As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system. We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.
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影响因子：
4.5
作者：
Cai, XZ;Hagedorn, CH;Cullen, BR
通讯作者：
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DOI：
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发表时间：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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