Use of a Dicistronic Expression Cassette Encoding the Green Fluorescent Protein for the Screening and Selection of Cells Expressing Inducible Gene Products

To facilitate the screening and selection of cells expressing inducible gene products, we have constructed a plasmid that, by the inclusion of a viral internal ribosome entry site, permits the synthesis of a dicistronic mRNA encoding both a gene of interest and the gene encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. This greatly simplifies the task of clone selection, since GFP fluorescence can be visualized non-obtrusively in live cells with a standard fluorescence microscope. We have applied this method to the tetracycline-regulated expression system in which the expression of a target gene, placed under the control of a promoter containing the tetracycline operator sequence (tetO), can be induced by a tetracycline-regulated trans-activator protein (tTA). Binding of the tTA to the tetO is inhibited in the presence of tetracycline. Optimal results with this system require two sequential rounds of transfection and screening. Obtaining a cell line expressing high levels of functional tTA is greatly simplified by transiently transfecting a plasmid encoding GFP into a pool of cells that has first been transfected with a tTA-expressor construct and selecting GFP-positive cells using a fluorescence-activated cell sorter. In the second step, the tTA cell line can then be stably transfected with a dicistronic expressor-GFP cassette. This method eliminates the task of characterizing cell lines by the standard method of examining levels of the exogenously expressed protein in cell extracts of individual clones.