Green fluorescent protein as a reporter in translational assays (original) (raw)
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The FASEB Journal, 2004
Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. However, it is not generally accepted that GFP can also be used as a quantitative reporter of gene expression. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured by flow cytometry. Two pieces of evidence support this conclusion: GFP fluorescence increases in direct proportion to the GFP gene copy number delivered to cells by a replication-defective adenovirus vector, Ad.CMV-GFP, and the intensity of GFP fluorescence is directly proportional to GFP mRNA abundance in cells. This conclusion is further supported by the fact that the induction of GFP gene expression from two inducible promoters (i.e., the TRE and ICP0 promoters) is readily detected by flow cytometric measurement of GFP fluorescent intensity. Collectively, the results presented herein indicate that GFP fluorescence is a reliable and quantitative reporter of underlying differences in gene expression.
A single secreted luciferase-based gene reporter assay
Analytical Biochemistry, 2014
Promoter analysis typically employs a reporter gene fused to a test promoter combined with a second reporter fused to a control promoter that is used for normalization purposes. However, this approach is not valid when experimental conditions affect the control promoter. We have developed and validated a single secreted luciferase reporter (SSLR) assay for promoter analysis that avoids the use of a control reporter. The approach uses an early level of expression of a secreted luciferase linked to a test promoter as an internal normalization control for subsequent analysis of the same promoter. Comparison of the SSLR assay with the dual luciferase reporter (DLR) assay using HMGCR (3-hydroxy-3-methylglutarylcoenzyme A reductase) and LDLR (low-density lipoprotein receptor) promoter constructs, which are down-regulated by 25-hydroxycholesterol, show that both assays yield similar results. Comparison of the response of the HMGCR promoter in SSLR transient assays compared very favorably with the response of the same promoter in the stable cell line. Overall, the SSLR assay proved to be a valid alternative to the DLR assay for certain applications and had significant advantages in that measurement of only one luciferase is required and monitoring can be continuous because cell lysis is not necessary.
Detection of promoter activity by flow cytometric analysis of GFP reporter expression
Nucleic Acids Research, 2002
Low ef®ciency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially dif®cult to ef®ciently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quan-ti®ed by¯ow cytometry. In this system, green¯uorescent protein (GFP) reporter constructs are cotransfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection ef®ciency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color¯uorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low ef®ciency.
Long-Term, Stable Expression of Green Fluorescent Protein in Mammalian Cells
Biochemical and Biophysical Research Communications, 1997
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,
Analytical Biochemistry, 2003
Efficient screening for ligands of seven-transmembrane, G-protein-coupled receptors, whether transfected or endogenously expressed, often involves cell-based reporter assays. Here we describe the development of reporter gene assays in HeLa cells. The reporter construct includes a synthetic multifunctional promoter with several different response motifs (NF-jB, STAT, and AP-1) and hence efficiently funnels several signaling pathways. The assay, performed with the resulting reporter cell line HFF11, has an exceptional high Z-factor and a large signal-to-background ratio. To facilitate cell handling during screening, we introduced a secreted Renilla luciferase as a reporter enzyme. HR36 reporter cells, equipped with the construct, were added to ligands present in a multiwell plate and after addition of coelenterazine they produced a luminescence readout. This procedure economizes cell handling and at the same time increases assay quality and sensitivity Ó