Navigation Links
New Reporter Plasmids for Studying Interferon-Stimulated Signal Transduction,,,Pathways


Monitor activation status of interferon-stimulated signaling pathways

Li Xu Tim Sanchez Mary Buchanan Chao-Feng Zheng
Stratagene

Stratagenes family of PathDetect cis-reporting systems now includes the GAS and ISRE reporting systems, to better study the activation of signal transduction pathways stimulated by interferon - a, - b, or - g. The pISRE-Luc cis-reporter plasmid contains direct repeats of the interferon-stimulated response element (ISRE) found in the promoter of the 54-kDa interferon-stimulated gene (ISG54); the pGAS-Luc cis-reporter plasmid contains direct repeats of the gamma-activating sequence from the promoter of the guanylate-binding protein. In these plasmids, a basic promoter element (TATA box), and the direct repeats of ISRE or GAS, control expression of the luciferase gene. The pISRE-Luc and pGAS-Luc reporter plasmids monitor the activation status of signaling molecules and transcription factors that mediate the function of interferons.

Interferons (IFNs) are soluble proteins that act as local hormones, inhibit viral replication, and activate certain host defense responses. These interesting proteins modulate the expression of many genes and elicit a wide range of biological functions, including tissue repair, activation of various blood cells involved in antibody production, inflammation, and host defense against viruses.2,3 IFNs have also been used as therapeutic agents against many viral diseases, including acquired immune-deficiency syndrome (AIDS).1,2,3

IFN-a and IFN-b are interferons synthesized by many cell types following viral infection and have two major functions: They inhibit viral replication by activating cellular genes whose products are involved in the destruction of viral RNA and in the inhibition of protein translation; and they induce expression of MHC class I molecules by most cells in the body, except virus-infected cells. In contrast, type II interferon (IFN-g) is produced mainly by effector T cells after the induction of the adaptive immune response.2,3

The actions of interferons are mediated by members of the class II cytokine receptor family. Stimulation of cells with IFNs results in the dimerization of their receptors and the activation of the associated protein tyrosine kinases, including JAKs and Tyks. These events lead to the activation and nuclear localization of a group of transcription factors known as signal transducers and activators of transcription (STATs).3 STATs can then bind to cis-acting DNA elements present in the promoters of various IFN-inducible genes and modulate their expression.3

At this time, six different STATs have been cloned and characterized from mammalian cells. These STATs can form homo- and heterodimers and bind to very similar yet different symmetrical, dyad sequences.3 When cells are stimulated with IFN-a or IFN-b, a DNA-binding complex is formed that consists of STAT1, STAT2, and another protein known as p48. This complex binds to the enhancer element, IFN-stimulated response element (ISRE). In contrast, the STAT1 homodimer, which binds to the unique gamma-activated sequence (GAS), is formed in response to IFN-g.3 The mechanism of the activation and nuclear localization of STATs is still not fully understood. To allow interferon-related regulation of transcription to be better characterized, Stratagene has introduced two new PathDetect cis-reporting systems that use the cis-acting enhancer elements ISRE and GAS.

PathDetect Cis-Reporting Systems

All PathDetect cis-reporting systems feature a cis-reporter plasmid that contains the luciferase reporter gene driven by the basic promoter element (TATA box) joined to direct repeats of cis-acting DNA elements.4 When a plasmid expressing a gene of interest is cotransfected into mammalian cells with a cis-reporter plasmid, increased luciferase expression indicates either direct or indirect activation of the transcription factors that bind to the cis-acting elements in the plasmids. The cis-reporting plasmids can be used to evaluate the effect of uncharacterized genes, growth factors, drug candidates, or the effects of extracellular stimuli on these transcriptional elements.

Construction and Testing of the pISRE-Luc Plasmid

The expression of a group of human genes, the interferon-stimulated genes (ISGs), is induced by IFN-a and IFN-b, the type I interferons.5,6 One of these genes, termed ISG54, because it encodes a 54-kDa protein, has a cis-acting element (TAGTTTCACTTTCCC, nucleotides -101 to -87) in its promoter.7 This element is responsible for the inducible expression of the ISG54 gene and is referred to as IFN-stimulated response element (ISRE).7 To create the pISRE-Luc plasmid, we inserted five direct repeats of this ISRE upstream of the basic promoter element (TATA box) and luciferase gene of the PathDetect cis-reporter plasmid backbone.

Fig.1

To test the specificity of the pISRE-Luc plasmid, we analyzed the response of the synthetic promoter by transfecting the plasmid into HeLa cells, then treating cells with increasing amounts of IFN-a (Figure 1, solid squares) and IFN-b (Figure 1, solid circles). For H eLa cells transfected with the pISRE-Luc plasmid and treated with 200 to 10,000 U/ml of IFN-a 9- to 16-fold increase in luciferase activity was seen. As expected, IFN-b treatment of the cells also stimulated luciferase expression from the pISRE-Luc plasmid up to 11-fold (Figure 1, solid circles). Although the pISRE-Luc plasmid was somewhat responsive to IFN-g (Figure 1, solid triangles), the luciferase activity was considerably higher for HeLa cells treated with IFN-a and IFN-b (Figure 1, solid squares and solid circles). This weak response of the pISRE-Luc plasmid to IFN-g could be attributed to the similarity of the ISRE and GAS sequences; it is also likely that types I and II interferons use a few common components to transmit their signals to the cell nucleus. However, neither IFN-a nor IFN-b induced luciferase expression from HeLa cells transfected with the pCIS-CK plasmid, a control plasmid that differs from the cis-reporter plasmids only in that it lacks an enhancer sequence (Figure 1, open squares and open circles). We conclude that the pISRE-Luc reporter plasmid is effective for monitoring the signaling pathways activated by type I interferons.

Construction and Testing of the pGAS-Luc Plasmid

The human gene encoding guanylate-binding protein (GBP) is induced in fibroblasts by INF-g within 15 minutes of treatment. An IFN gamma-activating sequence (GAS) has been identified in the GBP promoter (nucleotides -123 to -103).1 To construct the pGAS-Luc plasmid, we inserted four direct repeats of this GAS enhancer sequence upstream of the TATA box and luciferase gene of the PathDetect cis-reporter plasmid backbone.4

Fig.2

To evaluate the pGAS-Luc plasmid, we performed a series of transfection experiments. For HeLa cells transfected with the pGAS-Luc plasmid, then treated with 20 to 1,000 U/ml of IFN-g (Figure 2, solid triangles), luciferase activity, increased 4- to 10-fold, as compared to cells transfected with the pGAS-Luc plasmid but receiving no interferon treatment (Figure 2, open circles). Neither IFN-a nor IFN-b treatment of HeLa cells transfected with pGAS-Luc increased luciferase expression significantly (Figure 2, solid squares and solid circles). IFN-g did not induce luciferase expression from HeLa cells transfected with the pCIS-CK control plasmid (Figure 2, open triangles). Therefore, the direct repeats of GAS in the pGAS-Luc plasmid are responsible for the inducible expression of luciferase, and the reporter plasmid pGAS-Luc can be used to monitor the signaling pathways activated by IFN-g.

Both the pISRE-Luc and pGAS-Luc plasmids functioned in other cell lines as well. Luciferase expression in Chinese hamster ovary (CHO) cells transfected with the pISRE-Luc plasmid, for example, increased by over 8-fold after treatment with 2,000 U/ml IFN-b (data not shown). However, due to the varying biochemical composition of cell lines, we expect that the luciferase levels resulting from transfection of these plasmids into different cell lines will also vary.

Conclusions

Stratagenes new pISRE-Luc and pGAS-Luc cis-reporter plasmid scan be used to monitor the activation of signal pathways by IFN-a, -b, or -g. Upon transfection of the pISRE-Luc plasmid into HeLa cells and subsequent treatment with IFN-a or IFN-b, a significant increase in luciferase activity was seen. Similarly, increased luciferase expression levels resulted from IFN-g treatment of HeLa cells transfected with the pGAS-Luc plasmid. Therefore, the activation status of signaling molecules and transcription factors can be monitored with these two new PathDetect cis-reporter plasmids.

REFERENCES
  1. Decker, T., et al. (1991) EMBO J. 10: 927-932.

  2. Wilks, A.F. and Harpur, A.G. (1994) BioEssays 16: 313-320.

  3. Ihle, J.N. (1996) Cell 84: 331-334.

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

  5. Larner, A.C., et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6733-6737.

  6. Larner, A.C., et al. (1986) J. Biol. Chem. 261: 453-459.

  7. Levy, D.E., et al. (1998) Genes Devel. 2: 383-393.


'"/>

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. Stable HeLa Luciferase Reporter Cell Lines Expressing GAL4 Fusion Transactivators
3. Versatile Reporter Vectors for Monitoring Viral Transduction
4. Genetic Reporter Systems
5. Genetic Reporter Systems
6. Detection of Reporter Gene Activity in Cell Cultures and Murine Epidermis After Helios Gene Gun-Mediated Particle Bombardment
7. 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)
8. Luciferase Reporter Gene Cells and Recombinant Luciferase Dilutions Studied with the CLIPR Luciferase Assay Kit and the LMax Microplate Luminometer (MaxLine Application Note #38)
9. Promegas Multiplexed Luciferase Reporter and Cell Viability Assays performed on the PHERAstar
10. New Fusion Trans-Activator Plasmids for Studying Signal Transduction Pathways
11. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:1/12/2017)... ... January 12, 2017 , ... Each year, Crain’s Detroit Business News ranks the ... evaluates the patent estate of a company, its impact and significance, and the likelihood ... the way in technologies that transform energy sources such as low dose X-ray and ...
(Date:1/12/2017)... ... January 12, 2017 , ... After ... Lisa Rosendahl’s doctors gave her only a few months to live. Now a ... that has stabilized Rosendahl’s disease and increased both the quantity and quality of ...
(Date:1/12/2017)... and Pune, India , January 12, 2017 ... Toxicity Testing Market by Type and End Users - Global Opportunity Analysis and Industry ... million by 2022 from $2,921 million in 2015, growing at a CAGR of 15.07% ... ... Allied Market Research Logo ...
(Date:1/11/2017)... Colo. (PRWEB) , ... January 11, 2017 , ... ... the journal Clinical Cancer Research show early promise of the investigational anti-cancer agent ... despite a median 5 previous treatment regimens. Twenty-seven percent of these heavily pretreated ...
Breaking Biology Technology:
(Date:1/6/2017)... -- Delta ID Inc., a leader in consumer-grade iris scanning ... CES® 2017. Delta ID has collaborated with Gentex Corporation ... of iris scanning as a secure, reliable and convenient ... car, and as a way to elevate the security ... ID and Gentex will demonstrate (booth #7326 LVCC) a ...
(Date:1/3/2017)... LAS VEGAS , Jan. 3, 2017 ... announced the introduction of Onitor Track, an innovative biometric ... and men, showcasing this month at the 2017 Consumer ... . In the U.S., the World ... affect more than two-thirds of adults who are overweight ...
(Date:12/20/2016)... , Dec. 20, 2016 The ... sharing, rental and leasing is stoking significant interest ... radio frequency technology, Bluetooth low energy (BLE), biometrics ... as the next wave of wireless technologies in ... access system to advanced access systems opens the ...
Breaking Biology News(10 mins):