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Gene Promoter Reporter Vector

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Gene Promoter Reporter Vector Gene Promoter Reporter Vector User Manual Cat #: LR1XXX P/N 16116 Rev. A 050108 DRAFT September 3, 2008 9:21 pm GenePromoterReporterVectorTitle.fm Panomics, Inc. Gene Promoter Vector User Manual Copyright © Copyright 2008, Panomics, Inc. All rights reserved. Trademarks All other trademarks are of their respective owners. Citing Panomics in Publications When describing a procedure for publication using this product, we would appreciate it if you would refer to it as the Gene Promoter Vector Kit from Panomics. If a paper cites our product and is published in a research journal, the lead author(s) may receive a travel stipend for use at a technology conference or tradeshow by sending a copy of the paper to our technical support group at [email protected] or via fax at (510) 818-2610. Disclaimer Panomics, Inc. reserves the right to change its products and services at any time to incorporate technological developments. This manual is subject to change without notice. Although this manual has been prepared with every precaution to ensure accuracy, Panomics, Inc. assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. DRAFT September 3, 2008 9:21 pm GenePromoterReporterVectorTitle.fm Introduction Introduction Introduction Eukaryotic gene expression is regulated by a wide variety of developmental and environmental stimuli. First, an extracellular signaling molecule binds to a specific receptor. The signal is then transmitted through a series of molecular cascades, which activate or deactivate specific transcription factors (TFs) that regulate gene expression. The expression of any given gene is controlled by multiple transcription factors, which in turn are modulated by multiple signal transduction pathways. These pathways are also interconnected by molecular “cross-talk” (1–2). Activated TFs associate with the 5’ flanking region (promoter region) of the genes and RNA polymerase-mediated transcription is subsequently initiated. With Panomics' Gene Promoter Reporter Vectors, you can monitor and even quantify the promoter activity of 34 genes. Each Gene Promoter Reporter Vector contains a ~1 kB insert, corresponding to the 5'-flanking sequence located approximately 800 bp upstream of exon 1 of a specific human gene and extending to the first 50-200 bp of the untranslated region in exon 1 (Fig. 1, top). This insert is placed upstream of the luciferase reporter gene. Since the putative cis-acting enhancer elements are expected to exist in this cloned promoter region, the luciferase activity observed during the reporter assay more closely resembles the actual regulation of these genes within human cells. Binding of transcription factors in this region results in the expression of firefly luciferase, an enzyme capable of catalyzing a powerful bioluminescent reaction. Light emitted from the chemical reaction is directly proportional to the amount of expressed enzyme and thus the promoter activity of each gene. The backbone of the vector contains an ampicillin resistance gene for cloning purposes, an origin of replication, and an f1 origin for single-stranded DNA production (Fig. 1, bottom). Currently, Gene Promoter Reporter Vectors are available for 34 human genes. See www.panomics.com for an up-to-date list of all of the Gene Promoter Reporter Vectors. As a means of measuring promoter response in cells, the luciferase assay is simple, straightforward, and very effective. The reporter vector is first transfected into cells. After a limited amount of time, the cells are lysed and the substrate of luciferase, luciferin, is introduced into the cellular extract along with Mg2+ and excess ATP. Under these conditions, luciferase enzyme expressed by the reporter vector will catalyze the oxidative chemical reaction, the product of which can be detected and quantified by a luminometer or scintillation counter. The amount of light detected from the cell lysate correlates directly with the promoter activity of each human gene. Gene Promoter Reporter Vectors facilitate the study of promoter regulation mediated gene expression. With the aid of the reporter vectors, in vivo promoter activity in cell lines of various origins or those treated with a stimulus of interest can easily be compared. Cotransfect a vector expressing a gene of interest along with a Gene Promoter Reporter Vector to observe the effects of the gene of interest on signaling pathway activity. Gene Promoter Reporter Vector User Manual Page 3 Introduction Kozaks consensus 5’ flanking regionUTR in exon 1 Luc ORF +1 ATG ~800 bp 50–200 bp NheI Gene Promoter f1 ori enhancer BglII Ampr Gene Reporter Vector (~6 kb) Luciferase pUC on SV40 polyA Fig. 1: (Top) Panomics Gene Promoter Reporter Vector contains approximately 800 bp of 5' flanking region and 50-200 bp of untranslated region of exon 1 of a specifc human gene.. This ~1 kb region is located upstream of the start codon of luciferase gene. (Bottom) The vector backbone contains an ampicillin-resistance gene which allows ampicillin selection in E. coli, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production.. All of the Gene Reporter Vectors contain the Panomics signature sequence. These vectors are intended for research use only and should not be used commercially. Page 4 Gene Promoter Reporter Vector User Manual Materials Materials Materials Provided Gene Promoter Reporter Vector 20 µl of 500 ng/µl; 10 µg Control Reporter Vector 20 µl of 500 ng/µl; 10 µg * Sequence information is available on our website. Additional Items Items Required but not provided Required Item Source Transfection reagent FuGENE™ 6 Transfection Reagent (Roche, Cat. # 1 815 019) Reduced serum culture media Opti-MEM™ Reduced Serum Medium (Invitrogen, Cat. # 31985-062) Standard cell culture supplies Major Laboratory Supplier Luciferase reagent Luciferase Assay System (Promega, cat.# E150C) Luminometer Panomics Luminometer or equivalent Transfection The procedure is modified for using FuGENE 6 (Roche) in a 12-well culture plate. We do not recommend the use of other transfection methods, as this leads to reduced luciferase activity from the reporter. Guidelines for transfecting Gene Promoter Reporter Vector with FuGENE 6 Culture Vessel Volume of Vector DNA (µg) FuGENE 6 and dilution volume Reagent plating medium 96-well 100 µL 0.05 µg in 5 µL 0.1 - 0.3 mL 24-well 500 µL 0.2 µg in 20 µL 0.6 - 1.8 mL 12-well 1 mL 1.0 µg in 50 µL 1.2 - 3.6 mL 35-mm 2 mL 2.0 µg in 100 µL 3.0 - 9.0 mL 6-well 2 mL 2.0 µg in 100 µL 3.0 - 9.0 mL 60-mm 5 mL 5 µg in 200 µL 6.0 -20.0 mL 5 1 The day before transfection, plate 1-3x10 cells in 1 ml of their normal growth medium containing serum without antibiotics. Always plate cells in duplicate. This amount of cells should yield 50–80% confluence on the day of transfection. Note: Lower confluence is required to allow enough surface area for growth during the experiment period. Gene Promoter Reporter Vector User Manual Page 5 Materials 2 For each well of cells to be transfected, dilute 0.5 µg (1 µl) of the Gene Pro- moter Reporter Vector or the Control Vector with 50 µl of Opti-MEM I Reduced Serum Medium or serum-free culture media. Note: Each vector should be trans- fected in duplicate; one group of cells is for treatment and the other group is for control. 3 For each well, add 3 µl FuGENE 6 Reagent directly into 50 µl of Opti-MEM I Reduced Serum Medium or serum-free culture media, mix, and incubate for 5 min at room temperature, but no longer. This dilution can be prepared in bulk for multiple wells. 4 Combine the diluted Gene Promoter Reporter Vector with the diluted FuGENE 6 Reagent and mix gently. Incubate for 15-45 min at room temperature to allow DNA/transfection reagent complexes to form. Note: Do not allow undiluted FuGENE 6 Reagent to come into contact with plastic surfaces other than pipette tips. Once the FuGENE 6 Reagent is diluted, combine it with the diluted DNA within 45 min. 5 Add 100 µL of the DNA/transfection reagent complex directly to the complete growth media on cells and mix gently by rocking the plate back and forth. 6 Incubate the cells at 37°C in a C02 incubator, overnight. Note: It is possible to allow the transfection reaction to proceed for only 6-8 hrs. However, we recommend allowing 12 - 16 hr for gene expression, prior to treat- ment of cells. 7 After transfection, the media should be removed and; for an untreated sample, replaced with new media; for a treated sample, replace old media with new media that has been treated with the stimulus. When removing and replacing media, use caution to ensure that the cells remain attached to the bottom of the plate. 8 Keep the transfected cells at 37°C in a C02 incubator for the remainder of the incubation period. Inducer concentrations and duration of stimulation should be optimized for cell type and inducer reagent used. Page 6 Gene Promoter Reporter Vector User Manual Materials Collecting cells 1 After incubation, aspirate to completely remove the media from the culture plates. Be careful not to disturb the cells in the process. 2 Lyse the attached cells by adding lysis buffer (Promega, Luciferase Assay Sys- tem) to each well. Use approximately 50 µL per well for a 12-well plate; 100 µL per well for a 6-well plate. 3 To detach cells from the plate, immediately pipet the mixture up and down. 4. Transfer the cell lysate/buffer solution to a clean 1.5-ml microcentrifuge tube. Keep on ice or store at -20°C.
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