Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum

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.