一系列用于重组克隆的盘基网柄菌表达载体。

A series of Dictyostelium expression vectors for recombination cloning.

作者信息

Thomason Peter A, Brazill Derrick T, Cox Edward C

机构信息

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

出版信息

Plasmid. 2006 Nov;56(3):145-52. doi: 10.1016/j.plasmid.2006.04.001. Epub 2006 Jun 12.

DOI:10.1016/j.plasmid.2006.04.001

PMID:16765443

Abstract

We describe a series of Dictyostelium expression vectors for recombination cloning using the Gateway technology. DNA fragments generated by high fidelity polymerase chain reaction are cloned by topoisomerase-mediated ligation, then recombined into any of several Dictyostelium expression vectors using phage lambda LR recombinase. No restriction enzymes are used in this procedure. Coding regions can be expressed from their own promoters, or from a strong actin 15 promoter as a native protein, or with an amino or carboxyl-terminal GFP fusion. Gene promoters of interest can be analyzed by controlled expression of GFP and beta-galactosidase. These vectors allow for rapid and simple characterization of novel DNA, and are ideal for high-throughput studies.

摘要

我们描述了一系列使用Gateway技术进行重组克隆的盘基网柄菌表达载体。通过高保真聚合酶链反应产生的DNA片段通过拓扑异构酶介导的连接进行克隆，然后使用噬菌体λ LR重组酶重组到几种盘基网柄菌表达载体中的任何一种中。此过程不使用限制性内切酶。编码区可以从其自身的启动子表达，或者从强肌动蛋白15启动子作为天然蛋白表达，或者与氨基或羧基末端的绿色荧光蛋白（GFP）融合表达。感兴趣的基因启动子可以通过GFP和β-半乳糖苷酶的可控表达进行分析。这些载体能够快速简单地表征新的DNA，是高通量研究的理想选择。