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 inducibl e gene products, we have constructed a plasmid that, by the inclusion of a viral internal ribosome entry site, permits the synthesis of a di cistronic mRNA encoding both a gene of interest and the gene encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victor ia. This greatly simplifies the task of clone selection, since GFP flu orescence can be visualized non-obtrusively in live cells with a stand ard fluorescence microscope. We have applied this method to the tetrac ycline-regulated expression system in which the expression of a target gene, placed under the control of a promoter containing the tetracycl ine operator sequence (tetO), can be induced by a tetracycline-regulat ed trans-activator protein (tTA). Binding of the tTA to the tetO is in hibited in the presence of tetracycline. Optimal results with this sys tem require two sequential rounds of transfection and screening. Obtai ning a cell line expressing high levels of functional tTA is greatly s implified by transiently transfecting a plasmid encoding GFP into a po ol of cells that has first been transfected with a tTA-expressor const ruct and selecting GFP-positive cells using a fluorescence-activated c ell sorter. In the second step, the tTA cell line can then be stably t ransfected with a dicistronic expressor-GFP cassette. This method elim inates the task of characterizing cell lines by the standard method of examining levels of the exogenously expressed protein in cell extract s of individual clones.