Long-Term, Stable Expression of Green Fluorescent Protein in Mammalian Cells (original) (raw)

1997, Biochemical and Biophysical Research Communications

temperature (up to 65ЊC), pH11, 1% SDS (sodium dode-Despite the proven utility of green fluorescent procyl sulphate), 6 M guanidinium chloride and remains tein (GFP) as a reporter molecule for transient gene resistant to most proteases for hours. Photobleaching expression, the adequacy of this marker for models of wild-type GFP is reportedly about half that of fluorequiring durable, high-level gene expression has rescein, and the protein's quantum yield is about 0.8. not been fully tested. To address this issue, we per-The utility of this protein in experimental biology is formed the transfection of Chinese Hamster Ovary being defined in a variety of cells. For example, GFP (CHO) cells with plasmid DNA encoding both GFP was used to track the cellular movements in the slime and neomycin phosphotransferase (neo) cassettes. mold Dictyostelium discoideum in real time (3). GFP The expression of GFP was measured after the cells fusion proteins like GFP-Lac I have been used to moniwere cultured in the presence or absence of G418tor chromosomal segregation in live bacteria (4). GFP mediated selective pressure. After removal of G418 has also been used to assess gene transfer techniques from the growth medium, the percentage of pooled (5, 6). While the merits of this marker are extensive, G418 resistant transfectants which co-expressed the some limitations have also been reported (7). GFP transgene increased or remained unchanged. Numerous publications (8-10) have proven the use-Flow cytometric and visual isolation of GFP-expressing cells was possible without continued selection in fulness of GFP as reporter molecule in the setting of G418. One cloned cell line, C463, maintained high-transient gene expression. However, it remains unclear level green fluorescence for 18 weeks in G418 and whether cell lines are able to maintain high-level GFP an additional 12 weeks in nonselective medium. Our expression over many passages in the absence of selecdata suggest expression of GFP does not confer a tive growth conditions. Here we demonstrate visual growth disadvantage in mammalian cells. ᭧ 1997 and flow cytometric isolation of mammalian cells which Academic Press maintain high-level GFP expression for months during and after removal of G418. MATERIALS AND METHODS Green fluorescent protein (GFP) is a convenient reporter molecule to monitor gene and protein expression Cell lines and transfections. Chinese Hamster Ovary (CHO) cells in a broad spectrum of model organisms (1). GFP is were grown in HAMS cell media supplemented with 10% fetal bovine able to produce green fluorescence when exited with a serum (FBS; Biofluids; Rockville, MD) and gentamicin 25mg/ml (Life blue light. No additional substrates are required to de-Technologies; Gaithersburg, MD). Cells were plated at 50% confluence in 100mm dishes. Calcium phosphate:DNA transfections were tect GFP and it can be monitored in live cells (e.g. performed according to the manufacturer's protocol (Promega; Madiprotein localization). Most GFPs currently in use are son, WI). 15mg of purified DNA (Qiagen; Chatsworth, CA) was used derived from the Pacific Northwest jellyfish, Aequorea for each transfection, and G418 (Life Technologies; Gaithersburg, victoria (2). Aequorea GFP is relatively small polypep-MD) was added to the cell media 48 hours post transfection at a final tide consisting of 238 amino acid residues. The purified, concentration of 0.7 mg/ml. properly folded GFP exhibits remarkable fluorescent Constructions of expression vectors. PCR amplification and TA stability toward different denaturants such as high cloning (Invitrogen, San Diego, CA) were used to replace the CD4 domain of pJM48 (11) with the commercially available EGFP gene (Clontech; Palo Alto, CA) and a stop codon. The resulting vector, pLCB38, encoded both EGFP and neomycin phosphotransferase 1 Address correspondence to J. L. Miller, Laboratory of Chemical Biology, Building 10, Room 9N308, National Institutes of Health, genes between adeno-associated virus inverted terminal repeats (AAV ITRs). For comparison, a plasmid without AAV ITR regions,