Department of Genetics, University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany.
Gene. 2010 Oct 1;465(1-2):1-8. doi: 10.1016/j.gene.2010.06.010. Epub 2010 Jun 27.
We describe here a series of vectors for ectopic expression of tagged proteins in Dictyostelium discoideum. These vectors allow the addition of N- or C-terminal tags (GFP, mRFP, 3xFLAG, 3xHA, 6xMYC or TAP) with an optimised polylinker sequence and no additional amino acid residues at the N- or C-terminus of the protein. The expression cassettes were introduced into vectors containing Blasticidin or Geneticin resistance markers and into integrating as well as extrachromosomal plasmids. The vectors are designed as high and low copy versions and thus allow for a limited expression level control. They are also convenient with regard to complementation, co- and super-transformation. Finally the vectors share standardised cloning sites, so that a gene of interest can be easily transferred between vectors as experimental requirements evolve. These vectors were used to study the localisation of several putative RNA processing proteins including EriA and DicerB.
我们在这里描述了一系列用于在盘基网柄菌中异位表达标记蛋白的载体。这些载体允许在优化的多克隆位点序列中添加 N 端或 C 端标签（GFP、mRFP、3xFLAG、3xHA、6xMYC 或 TAP），并且在蛋白质的 N 端或 C 端不添加额外的氨基酸残基。表达盒被引入含有博来霉素或遗传霉素抗性标记的载体中，并整合到整合和染色体外质粒中。这些载体设计为高拷贝和低拷贝版本，因此允许对表达水平进行有限的控制。它们在补正、共转化和超转化方面也很方便。最后，这些载体共享标准化的克隆位点，因此，随着实验要求的发展，可以很容易地在载体之间转移感兴趣的基因。这些载体被用于研究包括 EriA 和 DicerB 在内的几个假定的 RNA 加工蛋白的定位。
