Construction of a series of pCS2+ backbone-based Gateway vectors for overexpressing various tagged proteins in vertebrates.
Construction of a series of pCS2+ backbone-based Gateway vectors for overexpressing various tagged proteins in vertebrates.
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DOI：
10.1093/abbs/gmw107
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发表时间：
2016-12
影响因子：
3.7
通讯作者：
Hong-Yan Wang;Yang Li;T. Xue;Ningyan Cheng;Hai-Ning Du
中科院分区：
文献类型：
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作者：
Hong-Yan Wang;Yang Li;T. Xue;Ningyan Cheng;Hai-Ning Du
门户载体已被广泛开发，以促进许多物种的基因克隆；然而，缺乏一个与多种生物兼容的通用系统。pCS2+骨架表达质粒作为一种多用途表达载体，在哺乳动物/禽类培养细胞或爪蟾/斑马鱼胚胎中广泛用于信使rna （mrna）或蛋白的高水平表达。目前，还没有一套适合Gateway克隆系统的pCS2+骨干载体。在这里，我们生成了一组Gateway目的载体，命名为pGCS （pCS2+中的Gateway质粒）载体，它可以融合到常用的氨基或羧基末端标签，包括MYC， HA， FLAG, His， GST以及eGFP荧光表位。与传统的“消化-连接”克隆方法相比，这套基于pCS2+骨干的Gateway目标载体的系统生成允许体外DNA重组，具有高速度，准确性和可靠性。因此，我们的系统加速了功能蛋白的产生，这些功能蛋白可以在多种脊椎动物生物体中广泛表达，而无需将基因繁琐地转移到不同的表达载体中。此外，我们通过非营利性的Addgene质粒库将这一系列的网关载体提供给研究社区。
Gateway vectors have been extensively developed to facilitate gene cloning in numerous species; however, a universal system that is compatible for multiple organisms was lacking. As a multipurpose expression vector, pCS2+ backbone-based expression plasmids are widely used for high-level expression of messenger RNAs (mRNAs) or proteins in mammalian/avian culture cells or Xenopus/zebrafish embryos. To date, a suite of vectors with pCS2+ backbone applicable for Gateway cloning system were unavailable yet. Here, we generated a set of Gateway destination vectors, named as pGCS (plasmids of Gateway in pCS2+) vectors, which can be fused to a choice of frequently used amino- or carboxyl-terminal tags, including MYC, HA, FLAG, His, GST, as well as eGFP fluorescent epitope. The systematic generation of this set of pCS2+ backbone-based Gateway destination vectors allows for in vitro recombination of DNA with high speed, accuracy, and reliability compared with the traditional 'digestion-ligation' cloning approach. Thus, our system accelerates the production of functional proteins, which could be widely expressed in a large variety of vertebrate organisms without tediously transferring genes into different expression vectors. Moreover, we make this series of Gateway vectors available to the research community via the non-profit Addgene Plasmid Repository.