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Pgl4.74 tk renilla luciferase

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The PGL4.74 TK-Renilla-luciferase is a plasmid vector that expresses the Renilla luciferase reporter gene under the control of the herpes simplex virus thymidine kinase (HSV-TK) promoter. The Renilla luciferase enzyme catalyzes the oxidation of coelenterazine, resulting in the emission of light. This plasmid can be used for monitoring gene expression or reporter assays in various cell lines.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Dual luciferase kit, by Promega (2 mentions) Passive lysis buffer (plb), by Promega (2 mentions) Synergy ht plate reader, by Agilent Technologies (2 mentions) In fusion cloning, by Takara Bio (2 mentions) Nhei hindiii, by New England Biolabs (2 mentions)

Spelling variants of this product

Renilla luciferase (170 citations) PGL4.74 Renilla luciferase (4 citations) Renilla-TK (4 citations) Renilla luciferase pGL4.74 [hRluc/TK] plasmid (2 citations)

4 protocols using pgl4.74 tk renilla luciferase

1

Characterizing Rev-erbα Transcriptional Activity

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C2C12 cells were transfected with E4bp4-luciferase and pGL4.74 TK-Renilla-luciferase (Promega) constructs alongside 3XFlag-CMV-7.1-Rev-erbα49 (link) and pcDNA3.1-Ds-Red (control) plasmids using Lipofectamine 3000 (Thermo Fisher Scientific). The medium was changed 6 hours later, 24 hours after medium change cells were harvested in passive lysis buffer (Promega) and assessed for luciferase activity using a Dual Luciferase kit (Promega) on a Synergy HT plate reader (Biotek). Firefly luciferase signal was normalized to Renilla luciferase signal as a control for transfection efficiency.
Dierickx P., Zhu K., Carpenter B.J., Jiang C., Vermunt M.W., Xiao Y., Luongo T.S., Yamamoto T., Martí-Pàmies Í., Mia S., Latimer M., Diwan A., Zhao J., Hauck A.K., Krusen B., Nguyen H.C., Blobel G.A., Kelly D.P., Pei L., Baur J.A., Young M.E, & Lazar M.A. (2021). Circadian REV-ERBs repress E4bp4 to activate NAMPT-dependent NAD+ biosynthesis and sustain cardiac function. Nature cardiovascular research, 1(1), 45-58.
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2

E4bp4 promoter reporter assay

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An 835bp mouse E4bp4 promoter region containing the REV-ERB binding site was amplified from mouse genomic DNA and cloned into a promoterless pGL4.21 (Promega) reporter via restriction digestion with NheI/HindIII (NEB) and subsequent ligation with T4 ligase. DNA sequence was confirmed by Sanger sequencing. The primer sequences used for cloning are: NheI site-flanked fw: 5’-ggttctattggcaacaggtag-3’ and HindIII site-flanked rv: 5’-tcaaagcagctaccttaaggtg-3’. Mutated-REV-RE and deleted REV-RE containing versions of this plasmid were generated by NheI/HindIII restriction digestion of pGL4.21, followed by In-Fusion cloning (Takara) using the following primers: fw; 5’- ACCTGAGCTCGCTAGCGG-3’, rv; 5’-TGTGTTAGTAaaaaAGTTCCGAGCGCCGC-3, fw; 5’-GCTCGGAACTttttTACTAACACACATCTCTCGGCG-3’, rv; 5’-CCGGATTGCCAAGCTTCAAAG-3’, rv; 5’-TTAGTGTTCCGAGCGCCGCGCTA-3’, fw; 5’- CGCTCGGAACACTAACACACATCTCTCGGCGC-3’.
E4bp4-luciferase, pGL4.74 TK-Renilla-luciferase (Promega), 3XFlag-CMV-7.1-Rev-erbα49 (link) and pcDNA3.1 Ds-Red (control) plasmids were transfected using Lipofectamine 3000 (Thermo Fisher Scientific). The medium was changed after 6 hours and 24 hours after medium change cells were harvested.
Dierickx P., Zhu K., Carpenter B.J., Jiang C., Vermunt M.W., Xiao Y., Luongo T.S., Yamamoto T., Martí-Pàmies Í., Mia S., Latimer M., Diwan A., Zhao J., Hauck A.K., Krusen B., Nguyen H.C., Blobel G.A., Kelly D.P., Pei L., Baur J.A., Young M.E, & Lazar M.A. (2021). Circadian REV-ERBs repress E4bp4 to activate NAMPT-dependent NAD+ biosynthesis and sustain cardiac function. Nature cardiovascular research, 1(1), 45-58.
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3

Characterizing Rev-erbα Transcriptional Activity

Check if the same lab product or an alternative is used in the 5 most similar protocols
C2C12 cells were transfected with E4bp4-luciferase and pGL4.74 TK-Renilla-luciferase (Promega) constructs alongside 3XFlag-CMV-7.1-Rev-erbα49 (link) and pcDNA3.1-Ds-Red (control) plasmids using Lipofectamine 3000 (Thermo Fisher Scientific). The medium was changed 6 hours later, 24 hours after medium change cells were harvested in passive lysis buffer (Promega) and assessed for luciferase activity using a Dual Luciferase kit (Promega) on a Synergy HT plate reader (Biotek). Firefly luciferase signal was normalized to Renilla luciferase signal as a control for transfection efficiency.
Dierickx P., Zhu K., Carpenter B.J., Jiang C., Vermunt M.W., Xiao Y., Luongo T.S., Yamamoto T., Martí-Pàmies Í., Mia S., Latimer M., Diwan A., Zhao J., Hauck A.K., Krusen B., Nguyen H.C., Blobel G.A., Kelly D.P., Pei L., Baur J.A., Young M.E, & Lazar M.A. (2021). Circadian REV-ERBs repress E4bp4 to activate NAMPT-dependent NAD+ biosynthesis and sustain cardiac function. Nature cardiovascular research, 1(1), 45-58.
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4

E4bp4 promoter reporter assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
An 835bp mouse E4bp4 promoter region containing the REV-ERB binding site was amplified from mouse genomic DNA and cloned into a promoterless pGL4.21 (Promega) reporter via restriction digestion with NheI/HindIII (NEB) and subsequent ligation with T4 ligase. DNA sequence was confirmed by Sanger sequencing. The primer sequences used for cloning are: NheI site-flanked fw: 5’-ggttctattggcaacaggtag-3’ and HindIII site-flanked rv: 5’-tcaaagcagctaccttaaggtg-3’. Mutated-REV-RE and deleted REV-RE containing versions of this plasmid were generated by NheI/HindIII restriction digestion of pGL4.21, followed by In-Fusion cloning (Takara) using the following primers: fw; 5’- ACCTGAGCTCGCTAGCGG-3’, rv; 5’-TGTGTTAGTAaaaaAGTTCCGAGCGCCGC-3, fw; 5’-GCTCGGAACTttttTACTAACACACATCTCTCGGCG-3’, rv; 5’-CCGGATTGCCAAGCTTCAAAG-3’, rv; 5’-TTAGTGTTCCGAGCGCCGCGCTA-3’, fw; 5’- CGCTCGGAACACTAACACACATCTCTCGGCGC-3’.
E4bp4-luciferase, pGL4.74 TK-Renilla-luciferase (Promega), 3XFlag-CMV-7.1-Rev-erbα49 (link) and pcDNA3.1 Ds-Red (control) plasmids were transfected using Lipofectamine 3000 (Thermo Fisher Scientific). The medium was changed after 6 hours and 24 hours after medium change cells were harvested.
Dierickx P., Zhu K., Carpenter B.J., Jiang C., Vermunt M.W., Xiao Y., Luongo T.S., Yamamoto T., Martí-Pàmies Í., Mia S., Latimer M., Diwan A., Zhao J., Hauck A.K., Krusen B., Nguyen H.C., Blobel G.A., Kelly D.P., Pei L., Baur J.A., Young M.E, & Lazar M.A. (2021). Circadian REV-ERBs repress E4bp4 to activate NAMPT-dependent NAD+ biosynthesis and sustain cardiac function. Nature cardiovascular research, 1(1), 45-58.
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