A Gateway MultiSite recombination cloning toolkit.
A Gateway MultiSite recombination cloning toolkit.
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网关多站点重组克隆工具包。
DOI：
10.1371/journal.pone.0024531
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Stowers RS
中科院分区：
文献类型：
--
作者：
Petersen LK;Stowers RS
DNA构建体的产生通常是进行生物学实验的限速步骤。由于效率和可靠性的提高，使用Gateway系统的单个DNA片段的扩增克隆提供了优于传统限制性酶克隆的进步。在这里，我们介绍了一系列用于两个、三个和四个片段Gateway MultiSite重组克隆的入口克隆和目的载体，其优点包括增加的灵活性和多功能性。与Gateway单片段克隆方法不同，Gateway单片段克隆方法通常将变异纳入模型系统特定的目的载体中，而我们的Gateway MultiSite克隆策略则将变异纳入了易于生成的与模型系统无关的条目克隆中。特别是，我们提出了含有GAL4，QF，UAS，QUAS，eGFP和mCherry等插入的入门克隆，并通过使用它们来产生表达克隆，包括各种trp离子通道家族成员的GAL4和QF驱动程序，UAS和QUAS兴奋性和抑制性光门控离子通道，以及QUAS红色和绿色荧光突触囊泡标记物，来证明它们在果蝇中的体内功能。因此，我们建立了一个入门工具包的模块化网关多站点入口克隆可能适用于任何模型系统。还建立了网关多站点克隆的入口克隆和目标载体清单（www.gatewaymultisite.org）。
The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduce a series of entry clones and a destination vector for use in two, three, and four fragment Gateway MultiSite recombination cloning whose advantages include increased flexibility and versatility. In contrast to Gateway single-fragment cloning approaches where variations are typically incorporated into model system-specific destination vectors, our Gateway MultiSite cloning strategy incorporates variations in easily generated entry clones that are model system-independent. In particular, we present entry clones containing insertions of GAL4, QF, UAS, QUAS, eGFP, and mCherry, among others, and demonstrate their in vivo functionality in Drosophila by using them to generate expression clones including GAL4 and QF drivers for various trp ion channel family members, UAS and QUAS excitatory and inhibitory light-gated ion channels, and QUAS red and green fluorescent synaptic vesicle markers. We thus establish a starter toolkit of modular Gateway MultiSite entry clones potentially adaptable to any model system. An inventory of entry clones and destination vectors for Gateway MultiSite cloning has also been established (www.gatewaymultisite.org).
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