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pGL4.49[luc2P/TCF-LEF RE/Hygro] Vector

by Promega
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  • 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 signaling pathways, we have vectors available that respond to the MAPK/JNK pathway with the pGL4.44[luc2P/AP1 RE/Hygro] Vector, Wnt pathway with the pGL4.49[luc2P/TCF-LEF RE/Hygro] Vector, cAMP/PKA pathway with the pGL4.29[luc2P/CRE/Hygro] Vector, Calcium/Calcineurin pathway with the pGL4.30[luc2P/NFAT-RE/Hygro] Vector, NF-?B pathway with the pGL4.32[luc2P/NF-?B-RE/Hygro] Vector, MAP/ERK pathway with the pGL4.33[luc2P/SRE/Hygro] Vector and RhoA pathway with the pGL4.34[luc2P/SRF-RE/Hygro] Vector.

    Investigate additional signaling pathways with vectors for stress pathways and cytokine pathways.

    Applications

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

    Sequence Information

    pGL4.29[luc2P/CRE/Hygro] Vector Accession Number DQ904461 and vector sequence text file.

    pGL4.30[luc2P/NFAT-RE/Hygro] Vector Accession Number DQ904462 and vector sequence text file.

    pGL4.32[luc2P/NF-?B-RE/Hygro] Vector Accession Number EU581860 and vector sequence text file.

    pGL4.33[luc2P/SRE/Hygro] Vector Accession Number FJ773212 and vector sequence text file.

    pGL4.34[luc2P/SRF-RE/Hygro] Vector Accession Number FJ773213 and vector sequence text file.

    pGL4.44[luc2P/AP1 RE/Hygro] Vector Accession Number JQ858516 and vector sequence text file.

    pGL4.49[luc2P/TCF-LEF RE/Hygro] Vector Accession Number JX099537 and vector sequence text file.

    Activator/Pathway Transcription Factor Binding Site Vector Name Example Data
    MAPK/JNK AP1 AP1 Response Element (AP1 RE) pGL4.44[luc2P/AP1 RE/Hygro]
    Representative data for pGL4.44[luc2P/AP1 RE/Hygro] Vector in HEK293 cells upon stimulation with PMA. HEK293 cells were transiently transfected with pGL4.44[luc2P/AP1 RE/Hygro] and pGL4.75[hRluc/CMV] Vectors and assayed in a 96-well format as indicated in the protocol after six hours stimulation with PMA. Firefly luciferase luminescence normalized to the Renilla luciferase control is shown, with error bars indicating the S.E.M. for five replicates. Luminescence was detected after addition of Dual-Glo® reagents, using a GloMax® 96 instrument with a 0.5 second integration time.
    cAMP/PKA CREB Cyclic AMP Response Element pGL4.29[luc2P/CRE/Hygro]
    High levels of reporter induction for cAMP Response Element containing vector in transient transfections. pGL4.29 Vector was transiently transfected into HEK293 cells. After 24 hours, reporter gene expression was induced by adding 100uM forskolin for 5 hours. Noninduced controls were treated with an equivalent amount of DMSO. Luciferase activity was measured using the Bright-Glo™ Luciferase Assay System (Cat.# E2610). Results will vary depending on the cell line and the transfection protocol used.
    NF-?B NF-?B Nuclear Factor ?B Response Element pGL4.32[luc2P/NF-?B-RE/Hygro]
    High levels of reporter induction for Nuclear Factor ?B (NF- ?B) Response Element containing vector in transient transfections. pGL4.32 Vector was transiently transfected into HEK293 cells. After 24 hours, reporter gene expression was induced by 20ng/ul of TNF? for 5 hours. Noninduced controls were treated with an equivalent amount of standard media. Luciferase activity was measured using the ONE-Glo™ Luciferase Assay System (Cat.# E6110). Results will vary depending on the cell line and the transfection protocol used.
    Calcium/Calcineurin NFAT Nuclear Factor of Activated T-Cells (NFAT) Response Element pGL4.30[luc2P/NFAT-RE/Hygro]

    High levels of reporter induction for Nuclear Factor of Activated T-cells (NFAT) Response Element containing vector in transient transfections. pGL4.30 Vector was transiently transfected into HEK293 cells. After 24 hours, reporter gene expression was induced by adding 1uM ionomycin plus 10ng/ml PMA for 17 hours. Noninduced controls were treated with an equivalent amount of DMSO. Luciferase activity was measured using the Bright-Glo™ Luciferase Assay System (Cat.# E2610). Results will vary depending on the cell line and the transfection protocol used.

    MAP/ERK Elk-1/SRF Serum Response Element pGL4.33[luc2P/SRE/Hygro]
    High levels of reporter induction for Serum Response Element (SRE) containing vector in transient transfections. pGL4.33 Vector was transiently transfected into HEK293 cells. Four hours after transfection, cells were changed into serum-starved media. After 24 hours following transfection, reporter gene expression was induced 20% FBS plus 10ng/ml PMA for 6 hours. Noninduced controls were treated with an equivalent amount of standard media. Luciferase activity was measured using the ONE-Glo™ Luciferase Assay System (Cat.# E6110). Results will vary depending on the cell line and the transfection protocol used.
    RhoA SRF Serum Response Factor Response Element pGL4.34[luc2P/SRF-RE/Hygro]
    High levels of reporter induction for Serum Response Factor Response Element (SRF-RE) containing vector in transient transfections. pGL4.34 Vector was transiently transfected into HEK293 cells. Four hours after transfection, cells were changed into serum-starved media. After 24 hours following transfection, reporter gene expression was induced 20% FBS for 6 hours. Noninduced controls were treated with an equivalent amount of standard media. Luciferase activity was measured using the ONE-Glo™ Luciferase Assay System (Cat.# E6110). Results will vary depending on the cell line and the transfection protocol used.
    Wnt TCF-LEF TCF-LEF Response Element (TCF-LEF RE) pGL4.49[luc2P/TCF-LEF RE/Hygro]
    Representative data for pGL4.49[luc2P/TCF-LEF RE/Hygro] Vector in HEK293 cells upon stimulation with mWnt3a or LiCl. HEK293 cells were transiently transfected with pGL4.49[luc2P/TCF-LEF RE/Hygro] and assayed in 96-well format after 8 hours stimulation with mWnt3a or LiCl 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.
    5257ML2
  • Protocols

    Complete Protocol

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    pGL4.43[luc2P/XRE/Hygro] Vector Protocol

    PDF (211 KB)

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    pGL4.49[luc2P/TCF-LEF/Hygro] Vector Protocol

    PDF (205 KB)

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    pGL4.29[luc2P/CRE/Hygro] Vector Protocol

    PDF (156 KB)

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    pGL4.30[luc2P/NFAT-RE/Hygro] Vector Protocol

    PDF (155 KB)

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    pGL4.32[luc2P/NF-?B-RE/Hygro] Vector Protocol

    PDF (182 KB)

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    pGL4.33[luc2P/SRE/Hygro] Vector Protocol

    PDF (157 KB)

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    pGL4.34[luc2P/SRF-RE/Hygro] Vector Protocol

    PDF (157 KB)

  • Certificate of Analysis

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    Storage Conditions

    -30C TO -10C

    Use Restrictions

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

  • Resources

    Citations