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

• Published in: BioTechniques Vol. 22; no. 1; pp. 150 - 161
• Main Authors: Mosser, D.D, Caron, A.W, Bourget, L, Jolicoeur, P, Massie, B
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.01.1997; Eaton
• Subjects: Animals; Biological and medical sciences; Biotechnology; Blotting, Western; Cell Line; Cell Separation - methods; Cloning, Molecular - methods; Diverse techniques; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Genetic engineering; Genetic technics; Methods. Procedures. Technologies; Microscopy, Fluorescence; Modification of gene expression level; Molecular and cellular biology; Plasmids; Scyphozoa; Screening; Antibiotic; Coelenterata; Regulation(control); Reporter gene; Selection; Cnidaria; Trans acting regulatory factor; Invertebrata; Gene expression; Hybrid gene; Tetracycline
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/97221rr02

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 . 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 ( , can be induced by a tetracycline-regulated trans-activator protein (tTA). Binding of the tTA to the O 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.