Levi S, Polyakov M, Egelhoff T T
Department of Physiology and Biophysics, Case Western Reserve School of Medicine, Cleveland, Ohio 44106-4970, USA.
Plasmid. 2000 Nov;44(3):231-8. doi: 10.1006/plas.2000.1487.
We have constructed expression vectors for Dictyostelium discoideum which encode a green fluorescent protein (GFP) sequence upstream of a multicloning site for introduction of sequences of interest. Insertion of cDNAs into the multicloning site results in expression of fusion protein bearing an amino- or carboxyl-terminal GFP tag which can be used for fluorescent localization studies in Dictyostelium cells. A parallel construct fuses a FLAG epitope tag at the amino terminus of expressed protein. Each fusion cartridge was placed either in a G418-resistance vector allowing transactivated Ddp2-based extrachromosomal replication or in a vector allowing autonomous Ddp1-based replication. Distinct differences in expression stability were observed in the two vector types. When GFP-expressing cells were analyzed by fluorescence microscopy, significant cell-to-cell variability in expression level was observed when expression was based on the Ddp2 vector, while less cell-to-cell variation in expression level was observed when the Ddp1 backbone was used for expression.
我们构建了用于盘基网柄菌的表达载体，该载体在多克隆位点上游编码绿色荧光蛋白（GFP）序列，用于引入感兴趣的序列。将cDNA插入多克隆位点会导致带有氨基或羧基末端GFP标签的融合蛋白表达，该融合蛋白可用于盘基网柄菌细胞中的荧光定位研究。一个平行构建体在表达蛋白的氨基末端融合了一个FLAG表位标签。每个融合盒被置于一个允许基于反式激活的Ddp2的染色体外复制的G418抗性载体中，或者置于一个允许基于自主的Ddp1的复制的载体中。在两种载体类型中观察到表达稳定性的明显差异。当通过荧光显微镜分析表达GFP的细胞时，基于Ddp2载体进行表达时，在表达水平上观察到显著的细胞间变异性，而当使用Ddp1骨架进行表达时，在表达水平上观察到的细胞间变异性较小。
