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pGL4.45[luc2P/ISRE/Hygro] Vector

by Promega
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  • Overview
  • Protocols
  • Specifications
  • Resources
  • Creating a cell line with an indicator of a functional signaling pathway is useful for deciphering the components in a signaling pathway. These tools are made by inserting multiple repeats of a response element upstream of a minimal promoter (minP). We have designed vectors that report the activity of a variety of pathways using the optimized luc2 firefly luciferase gene in the pGL4 backbone. The luc2 gene is engineered to remove most cryptic transcription factor binding sites and improve mammalian expression through codon optimization. The luc2P and RapidResponse™ genes are luc2 genes appended with degradation sequences to influence the cellular half-life of firefly luciferase. Proteins encoded by the RapidResponse™ genes respond more rapidly to stimuli but at the expense of signal intensity. The luc2P (1-hour half-life) gene responds more rapidly than luc2 (3-hour half-life) with moderate signal intensity.

    These response element vectors also have a hygromycin resistance selectable marker for use either in transient transfection experiments or for selection of a stable cell line. Some of these response element vectors also are available stable cell lines (GloResponse™ Cell Lines) to speed your research. For investigating the cytokine signaling pathway, we have vectors available include that respond to interferon-? (INF-?) with the pGL4.45[luc2P/ISRE/Hygro] Vector, interleukin 6 (IL-6) with the pGL4.47[luc2P/SIE/Hygro] Vector, transforming growth factor ? (TGF-?) with the pGL4.48[luc2P/SBE/Hygro] Vector and interleukin 3 (IL-3) with the pGL4.52[luc2P/STAT5RE/Hygro] Vector.

    Learn about other signaling pathway vectors for stress pathways and additional signaling pathways.

    Applications

    • Transcription regulation.
    • Virus-cell interactions.
    • Compound screening.
    • Post-translational modifications.
    • GPCR signaling.
    • Cell signaling.

    Sequence Information

    pGL4.45[luc2P/ISRE/Hygro] Vector GenBank® Accession Number JQ858514 and vector sequence text file.

    pGL4.47[luc2P/SIE/Hygro] Vector GenBank® Accession Number JQ858512 and vector sequence text file.

    pGL4.48[luc2P/SBE/Hygro] Vector GenBank® Accession Number JQ858517 and vector sequence text file.

    pGL4.52[luc2P/STAT5 RE/Hygro] Vector GenBank® Accession Number JX206457 and vector sequence text file.

    Activator/Pathway Transcription Factor Binding Site Vector Name Example Data
    INF-? STAT1:STAT2 Interferon Stimulated Response Element (ISRE) pGL4.45[luc2P/ISRE/Hygro]
    Representative data for pGL4.45[luc2P/ISRE/Hygro] in U2OS cells upon stimulation with IFN?. U2OS cells were transiently transfected with pGL4.45[luc2P/ISRE/Hygro] Vector and assayed in 96-well format after 16 hours stimulation with IFN? as indicated in the protocol. Firefly luciferase luminescence normalized to untreated cells is shown, with error bars indicating the S.E.M. for three replicates. Luminescence was detected after addition of ONE-Glo® Reagent, using a GloMax® Multi+ instrument with a 0.5 second integration time.
    TGF-? SMAD3:SMAD4 SMAD Binding Element (SBE) pGL4.48[luc2P/SBE/Hygro]
    Representative data for pGL4.48[luc2P/SBE/Hygro] Vector in HEK293 cells upon stimulation with hTGF-?1. HEK293 cells were transiently transfected with pGL4.48[luc2P/SBE/Hygro] Vector and assayed in 96-well format after three hours stimulation with hTGF-?1 as indicated in the protocol. Firefly luciferase luminescence normalized to untreated cells is shown, with error bars indicating S.E.M. for three replicates. Luminescence was detected after addition of ONE-Glo® Reagent, using a GloMax® 96 instrument with a 0.5 second integration time.
    IL6 STAT3:STAT3 sis-Inducible Element (SIE) pGL4.47[luc2P/SIE/Hygro]
    Representative data for pGL4.47[luc2P/SIE/Hygro] Vector in HEK293 cells upon stimulation with IL-6. HEK293 cells were transiently transfected with pGL4.47[luc2P/SIE/Hygro] Vector and assayed in 96-well format after 24 hours stimulation with IL-6 as indicated in the protocol. Firefly luciferase luminescence normalized to untreated cells is shown, with error bars indicating the S.E.M. for three replicates. Luminescence was detected after addition of ONE-Glo™ Reagent, using a GloMax® Multi+ instrument with a 0.5 second integration time.
    IL3 STAT5:STAT5 STAT5 Response Element pGL4.52[luc2P/STAT5 RE/Hygro]
    Representative data for pGL4.52[luc2P/STAT5 RE/Hygro] Vector in Ba/F3 cells upon stimulation with mIL-3. Ba/F3 cells were transiently transfected with pGL4.52[luc2P/STAT5 RE/Hygro] Vector and assayed in 96-well format after 4 hours stimulation with mIL-3 as indicated in the protocol. Firefly luciferase luminescence normalized to untreated cells is shown, with error bars indicating the S.E.M. for four replicates. Luminescence was detected after addition of ONE-Glo® Reagent, using a GloMax® Multi+ instrument with a 0.5-second integration time.
  • Protocols

    Complete Protocol

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    pGL4.47[luc2P/SIE/Hygro] Vector Protocol

    PDF (195 KB)

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    pGL4.48[luc2P/SBE/Hygro] Vector Protocol

    PDF (192 KB)

    Download PDF

    pGL4.52[luc2P/STAT5RE/Hygro] Vector Protocol

    PDF (208 KB)

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    pGL4.45[luc2P/ISRE/Hygro] Vector Protocol

    PDF (215 KB)

  • Certificate of Analysis

    Lookup Certificate of Analysis

    Storage Conditions

    -30C TO -10C

    Use Restrictions

    For Research Use Only. Not for Use in Diagnostic Procedures.