Navigation Links
Stable HeLa Luciferase Reporter Cell Lines Expressing GAL4 Fusion,,,Transactivators


Stable reporter cell lines for signal transduction pathway readout

Lisa Hexdall Li Xu Alan Greener Mary Buchanan John Bauer Chao-Feng Zheng
Stratagene

Stratagene now offers the PathDetect HLR-CREB, HLR-Elk1, and HLR-cJun, cell lines F for studying signal transduction pathways that converge at the transcription factors CREB, Elk1, and c-Jun. These stable HeLa cell lines feature constitutive expression of the GAL4-CREB, GAL4-Elk1, or GAL4-cJun fusion proteins, respectively. They also contain the firefly luciferase gene, which is controlled by a promoter that responds to GAL4 fusions, as does the HLR cell line that contains only the GAL4-responsive luciferase expression plasmid. When activated by phosphorylation, these fusion proteins bind to the promoter and induce luciferase expression. Therefore, luciferase activity in stable cell lines reflects the activation status of the fusion transactivator and, hence, the activation status of corresponding signaling pathways. The effects of extracellular stimuli on signaling pathways that converge at these activators can be studied directly, without transfection. The potential applications of these cell lines include studying signal transduction and transcription mechanisms and assessing protein-protein interactions in mammalian cells. In addition, these cell lines can potentially be adapted for high-throughput drug screening.

Regulation of gene transcription by extracellular signals is pivotal for controlling growth and differentiation. Because the transcription machinery of eukaryotic cells is located inside the nucleus, signals outside the cells need to be transmitted across the cytoplasmic and nuclear membranes as well as the cytoplasm. This process of signal transmission, better known as signal transduction, involves elaborate networks of signaling molecules (e.g., receptors, protein kinases, and phosphatases) organized in linear or parallel pathways.1,2 These signaling pathways receive diverse upstream signals that cause distinct downstream changes. Among the better-characterized signal transduction pathways are the cAMP-dependent protein kinase (PKA) pathway and the mitogen-activated protein kinase (MAPK) pathways. When activated by upstream signals, PKA or MAPK translocates into the nucleus and activates transcription factors, such as CREB and Elk1.3 Thus, the activation level of the transcription factors reflects the status of the respective kinases and upstream signaling molecules along the pathway.

To facilitate studies on the in vivo functions of gene products or extracellular stimuli, Stratagene developed the PathDetect trans-reporting systems using GAL4 fusion transactivators as pathway-specific sensors.4,5,6,7 In these systems, a fusion trans-activator plasmid that consists of the DNA binding domain of the yeast GAL4 (residues 1 to 147) protein8 and the activation domain of CREB, Elk1, or c-Jun is used to probe the activity of PKA, MAPK, and JNK, respectively.4-7,9,10 These fusion trans-activators bind to the promoter in the pFR-Luc reporter plasmid, which contains five direct repeats of the GAL4-binding element and controls luciferase expression.4 Either direct or indirect phosphorylation of the activation domain of a fusion trans-activator protein by an uncharacterized gene product activates transcription of the luciferase gene. Thus, expression levels of luciferase reflect the activation status of the respective kinases that define the signal transduction pathways.

To offer expanded capability for studying signal transduction and improved performance for adaptation to high-throughput applications, Stratagene has developed stable HeLa reporter cell lines that express the GAL4-CREB, GAL4-Elk1, or GAL4-cJun. In the HLR-CREB, HLR-Elk1, and HLR-cJun cell lines, luciferase activity reflects the activation status of the chimeric trans-activator protein and, therefore, the respective signaling pathways. By performing simple luciferase assays, researchers can directly evaluate the effects of extracellular stimuli on signaling pathways converging at CREB, Elk1, or c-Jun. Use of these cell lines eliminates transfections, thereby saving time while avoiding the variables that can be caused by fluctuations in transfection efficiencies. The HLR cell line can be used to assess protein-protein interactions in mammalian cells,11 while the HLR-CREB, HLR-Elk1, and HLR-cJun cell lines can be used to study transcription mechanisms. Another application for these cells includes adaptation for high-throughput drug screenings, since many components of signal transduction pathways are potential targets for drug development efforts.

Establishing Stable Reporter Cell Lines

Fig.1

To create stable reporter cell lines with the PathDetect trans-reporting systems, we tried several approaches. Figure 1 outlines our most successful protocol: We first transfected the pFR-Luc plasmid with a hygromycin expression cassette into HeLa cells. Through successive rounds of selection in antibiotic medium, hygromycin-resistant clones were isolated and tested with the PathDetect CREB, Elk1, and c-Jun trans-reporting systems. Cell lysates from these stable clones were then prepared, assayed for luciferase activity, and analyzed. One clonal line, designated as HLR for the HeLa luciferase reporter cell line, was chosen for further experimentation. It gave the highest levels of luciferase activity when tested with the trans-reporting systems mentioned above and had the lowest background of luciferase activity.

Next, in separate experiments, we transfected the HLR cell line with the fusion trans-activator pFA2-CREB, pFA2-Elk1, or pFA2-cJun plasmids. After performing successive rounds of selection with G418, we chose the clones that gave the best response when transfected with the positive control vectors from the PathDetect kits. These clones were then subjected to more extensive analysis.

Testing Stable Cell Lines

Fig.2

Genomic DNA from each stable clone was prepared and used as a template for PCR analysis to confirm that each fusion trans-activator plasmid was stably incorporated into the genomes of the selected clones. To amplify the activation domain for each reporter cell line, we used one primer specific to the GAL4 DNA-binding domain and another primer specific to each of the activation domains. Each primer set successfully amplified a product of the expected size. Analysis of HLR-CREB and HLR-Elk1 cell lines are shown in Figure 2. To further confirm the activity and specificity of these cell lines, we transfected each cell line with plasmids encoding known activators of each pathway. Upon luciferase assay of cell lysates, each cell line showed significant luciferase expression above background (Figure 3).

Fig.3

Additional testing included an analysis of the activation of luciferase activity in these stable cell lines by extracellular stimuli. For example, HLR-Elk1 cells were treated with known MAPK activators, including epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) (Figure 4). Luciferase expression in the HLR-Elk1 cells was five times higher than the control, when cells were treated with 100 ng/ml of EGF, and almost 50-fold higher when cells were challenged with PMA (60 ng/ml). Dulbeccos modified Eagles medium (DMEM) and dimethylsulfoxide (DMSO) were used as negative controls.

Fig.4

For the final testing, we confirmed purity by screening for mycoplasma and established viability after freezing in liquid nitrogen. The HLR, HLR-CREB, HLR-Elk1, and HLR-cJun cell lines are available as frozen stocks containing 5 x 105 cells; each cell line includes the corresponding positive and negative control plasmids.

Conclusions

The HLR-CREB, HLR-Elk1, and HLR-cJun stable cell lines offer a new approach and added convenience for studying the intricate interactions of signal transduction pathways. These cell lines can be used to evaluate whether a new gene product or extracellular stimulus is involved in signaling pathways that converge at CREB, Elk1, and c-Jun transcription factors; furthermore, the cell lines can be used to identify which step along the pathway is targeted. The HLR-CREB, HLR-Elk1, and HLR-cJun cell lines can be adapted for high-throughput screening of inhibitors or activators of these signaling pathways. In addition, the HLR cell line can be used in a variety of other applications, such as assessing protein-protein interactions in GAL4-derived mammalian two-hybrid systems.

REFERENCES
  1. Boulikas, T. (1995) Critical Rev. Eukaryotic Gene Exp. 5: 1-77.

  2. Karin, M. and Hunter, T. (1995) Curr. Biol. 5: 747-757.

  3. Treisman, R. (1996) Curr. Opin. Cell Biol. 8: 205-215.

  4. Xu, L., et al. (1997) Strategies 10: 1-3.

  5. Xu, L., et al. (1997) Strategies 10: 79-80.

  6. Xu, L. and Zheng, C.-F. (1997) Strategies 10: 81-83.

  7. Sanchez, T., et al. (1998) Strategies 11: 52-53.

  8. Sadowski, I. and Ptashne, M. (1989) Nucl. Acid Res. 17: 7539.

  9. Marais R., et al. (1993) Cell 73: 381-393.

  10. Smeal, T., et al. (1994) EMBO J. 13: 6006-6010.

  11. Dang, C.V., et al. (1991) Mol. Cell. Biol. 11: 945-962.


'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence Humanized Renilla GFP Reporter
2. Dual-Luciferase Quantitation in the LMax Microplate Luminometer (MaxLine Application Note #39)
3. Luciferase Reporter Gene Cells and Recombinant Luciferase Dilutions Studied with the CLIPR Luciferase Assay Kit and the LMax Microplate Luminometer (MaxLine Application Note #38)
4. Promegas Multiplexed Luciferase Reporter and Cell Viability Assays performed on the PHERAstar
5. Versatile Reporter Vectors for Monitoring Viral Transduction
6. New Reporter Plasmids for Studying Interferon-Stimulated Signal Transduction Pathways
7. Genetic Reporter Systems
8. Genetic Reporter Systems
9. Detection of Reporter Gene Activity in Cell Cultures and Murine Epidermis After Helios Gene Gun-Mediated Particle Bombardment
10. A Microtiter-based Assay for the Determination of ID50s of b-lactamase Inhibitors Employing Reporter Substrates Detected at UV or Visible Wavelengths (MaxLine Application Note #20)
11. A Comparison Study of Lipid Tranfection Reagents in A549, NIH 3T3 and COS-7 Cell Lines
Post Your Comments:
(Date:7/29/2015)... , July 30, 2015 According ... Market - Growth, Trends & Forecasts (2015-2020), , published by ... US$ 30.25 billion by the end of 2020, with ... accounting for more than 40% of the global market size. ... at a CAGR of 13.7% during the period of (2015-2020). ...
(Date:7/29/2015)... - BioAmber Inc. (NYSE: BIOA ) today announced that ... has resigned.  The Company has hired Andrew Ashworth ... December 2014, as full time interim CFO and has ... Andy Ashworth began his employment today, to ... Andy was the Company,s CFO from September 2011 to ...
(Date:7/29/2015)... ... July 29, 2015 , ... G-CON Manufacturing, Inc., the ... their 2nd generation cell therapy POD® design. The 2nd generation portfolio not only ... a new POD® design. , “G-CON first offered our miniPOD CT product ...
(Date:7/29/2015)... 2015  AmnioChor Inc., an early stage biotech startup ... to announce that the Musculoskeletal Transplant Foundation of ... in their seed round of development of the proprietary ... AmnioChor,s technology allows cryopreservation of the ... cells living within those tissues. Amnion is a well-established ...
Breaking Biology Technology:Asia Pacific to Dominate the Global Stem Cell Market - Vericel Corporation, Ocata Therpeutics, Athersys Inc., Cryo-Cell International, Geron Corporation, StemCells, Inc. Among the Major Players 2Asia Pacific to Dominate the Global Stem Cell Market - Vericel Corporation, Ocata Therpeutics, Athersys Inc., Cryo-Cell International, Geron Corporation, StemCells, Inc. Among the Major Players 3BioAmber Announces Change in Chief Financial Officer 2BioAmber Announces Change in Chief Financial Officer 3G-CON Manufacturing Launches Their 2nd Generation Cell Therapy Cleanroom POD® 2G-CON Manufacturing Launches Their 2nd Generation Cell Therapy Cleanroom POD® 3The Musculoskeletal Transplant Foundation Agrees to Invest in AmnioChor 2
... - The Madison-based Imago Scientific Instruments has ... State University's College of Engineering for use in ... Analysis. , ,The lab will be the primary research ... led by Balaji Narasimhan, an associate professor of chemical ...
... Wisconsin's economy evolves, regional collaboration is key to the ... companies that do business here. , ,Our state boasts ... capital, but alone we lack the critical mass needed ... East and West Coasts - for the resources so ...
... CDW Corp. , the Illinois-based technology provider that recently ... top salesman to handle Berbee's transition into the company. ... sales Steven Schuldt in charge of merging Berbee, a ... in Vernon Hills, Ill., acquired the privately-held Berbee on ...
Cached Biology Technology:Regional collaboration is key to state's future 2Regional collaboration is key to state's future 3
(Date:7/8/2015)... NEW YORK , July 8, 2015 /PRNewswire/ ... today announced BD & Guidepoint Mentor, a ... access to Guidepoint,s expert network services. ... cutting-edge technologies to improve healthcare delivery and outcomes and, ... each start-up entrepreneur will be able to directly engage ...
(Date:7/8/2015)... SPRINGS, Florida , July 8, 2015 ... constantly evolving as this summer,s must have products such as ... in demand as popularity for biometrics based devices continues to ... are NXT-ID, Inc. (NASDAQ: NXTD ), Google Inc. (NASDAQ: ... Inc. (NYSE: FIT ), GoPro, Inc. (NASDAQ: GPRO ...
(Date:7/7/2015)... , July 7, 2015  Based on its ... & Sullivan recognizes Credence ID, LLC with the ... Company of the Year Award. Credence ID has ... in its mission of offering enrollment and identification ... As Credence ID was formed by experts from ...
Breaking Biology News(10 mins):BD and Guidepoint Team to Connect Healthcare Start-up Companies With Business and Scientific Experts 2BD and Guidepoint Team to Connect Healthcare Start-up Companies With Business and Scientific Experts 3Consumers Flock to This Summer's Must Have Tech Gadgets as Latest Digital Wallets, Smart Technology & Wearable Products Continue to Grow in Popularity & Functionality 2Consumers Flock to This Summer's Must Have Tech Gadgets as Latest Digital Wallets, Smart Technology & Wearable Products Continue to Grow in Popularity & Functionality 3Consumers Flock to This Summer's Must Have Tech Gadgets as Latest Digital Wallets, Smart Technology & Wearable Products Continue to Grow in Popularity & Functionality 4Consumers Flock to This Summer's Must Have Tech Gadgets as Latest Digital Wallets, Smart Technology & Wearable Products Continue to Grow in Popularity & Functionality 5Consumers Flock to This Summer's Must Have Tech Gadgets as Latest Digital Wallets, Smart Technology & Wearable Products Continue to Grow in Popularity & Functionality 6Frost & Sullivan Acknowledges the Distinctive Customer-focused Attributes of Credence ID's Mobile Biometric Product Suite 2Frost & Sullivan Acknowledges the Distinctive Customer-focused Attributes of Credence ID's Mobile Biometric Product Suite 3Frost & Sullivan Acknowledges the Distinctive Customer-focused Attributes of Credence ID's Mobile Biometric Product Suite 4Frost & Sullivan Acknowledges the Distinctive Customer-focused Attributes of Credence ID's Mobile Biometric Product Suite 5
... Special Surgery researchers have found that statins may be ... from pregnancy complications caused by antiphospholipid syndrome (APS), according ... the body produces antibodies directed at phospholipids, the main ... study published in the October issue of the ...
... from the Herman B Wells Center for Pediatric Research at ... family of blood proteins that they hope holds a ... and adults. Their findings may one day help ... and other cancers of the blood. The researchers, led ...
... have created nanowire sensors coupled with simple microprocessor electronics ... used for point-of-care (POC) disease detection, according to a ... use activation of immune cells by highly specific antigens ... as the detector. When T cells are activated, they ...
Cached Biology News:Statins may prevent miscarriages 2Statins may prevent miscarriages 3On the trail of a targeted therapy for blood cancers 2Sensitive nanowire disease detectors made by Yale scientists 2
... Service, We deliversoluble protein or No ... yields the best soluble protein results , ... clone submitted , Powerful results on your ... Solubility Optimization Service (SOS) uses proprietary tools ...
... The Rapid-Screen cDNA Library Panels were ... just three sets of PCRs to identify ... analysis is performed in a 96-well "Master ... from 5,000 clones. Having identified the positive ...
... ProteinChip beta-amyloid Multipeptide Kit, designed for ... detecting a variety of amyloid peptides ... before. - Clinical neuroscience proteomics applications ... treatment - Correlate beta-amyloid peptide levels ...
Formula variant: With the macro- and micronutrients as described by Lloyd and McCown (1981). Preparation Quantity Equivalency: Formulated to contain 2.3 grams of powder per liter of medium. Physical ...
Biology Products: