Hygromycin B-selected cell lines from GAL4-regulated pUAST constructs

Here we report the generation of stable, selectable Drosophila S2 cell lines using the UAS-GAL4 system. Cloning of the hygromycin resistance gene into the pUAST vector and cotransfection with other pUAST constructs in S2 cells results in coexpression of up to four different proteins under hygromycin selection. Protein expression is driven by the ubiquitous Actin5C-GAL4 driver and cell cultures are maintained in hygromycin-supplemented, serum-free media to ensure constitutive protein production. Visual comparison of cells cotransfected with GFP and RFP demonstrates a uniform cell population expressing both markers simultaneously, while Western blot analysis shows concurrent expression of MYC3-tagged proteins. In addition, fluorescent cell sorting (FACS) analysis shows that 80% of the total cell population express the GFP marker. Our data indicate that using this technique it is possible to establish stable, selectable cell lines that provide a pool of readily accessible protein. This facilitates protein-based studies and abolishes the need to carry out time-consuming and expensive transfections.