Pgl3 basic luciferase reporter vector

Manufactured by Promega

Sourced in United States, China

The PGL3-basic luciferase reporter vector is a plasmid designed for the expression and detection of luciferase activity. It provides a basic backbone for the construction of luciferase reporter gene constructs.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (44 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (19 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (13 mentions) Dual luciferase assay, by Promega (8 mentions) Dual luciferase reporter assay kit, by Promega (7 mentions) Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (6 mentions) Dual luciferase assay system, by Promega (6 mentions) Dual luciferase kit, by Promega (6 mentions) Luciferase assay system, by Promega (6 mentions) Prl tk, by Promega (5 mentions) Passive lysis buffer (plb), by Promega (4 mentions) Psicheck 2 vector, by Promega (4 mentions) Dual luciferase assay kit, by Promega (3 mentions) Prl tk renilla luciferase reporter plasmid, by Promega (3 mentions) Pcdna3.1 vector, by Thermo Fisher Scientific (3 mentions) Luciferase assay kit, by Promega (3 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (3 mentions) Dual glo luciferase assay system, by Promega (3 mentions) Dual luciferase reporter assay, by Promega (3 mentions) Pjet1.2 blunt cloning vector, by Thermo Fisher Scientific (3 mentions)

189 protocols using pgl3 basic luciferase reporter vector

Zebrafish p53 Promoter Activity Assay

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Based on zebrafish p53 DNA sequences (ENSDARG00000035559), the 2045 bp of p53 promoter region (nt −1214 to +831 bp) were cloned by PCR with the primers PGL3-p53-F/PGL3-p53-R and inserted into pGL3-basic luciferase reporter vector (Promega). A bioinformatics tool ConSite (http://consite.genereg.net/cgi-bin/consite) was used to predict transcription factor binding sites. The primer sequences are listed in Supplementary Table 1.
To validate the activity of p53 promoter, 2.5 × 10⁵ EPC cells seeded in 24-well plates overnight were transfected with 25 ng Renilla (Promega) and 500 ng p53 promoter or pGL3-basic luciferase reporter vector. After 48 h, the cells were collected and lysed. The luciferase activity of lysates was assayed with the Dual-Luciferase reporter assay kit (Promega) by a Junior LB9509 luminometer (Berthold, Pforzheim, Germany). To determine the effect of CD44a variants on the p53 promoter activation, 2.5 × 10⁵ EPC cells seeded in 24-well plates overnight were transfected with 250 ng p53 promoter and 25 ng Renilla, together with 250 ng p3×FLAG, CD44a_tv1-FLAG or CD44a_tv2-FLAG. The transfected cells were infected with E. piscicida at an MOI of 0.1 or left untreated. At 6 hpi, the cells were lysed. The luciferase activity of lysates were measured with the Dual-luciferase Reporter Assay System.

Cao L., Fang H., Yan D., Wu X.M., Zhang J, & Chang M.X. (2022). CD44a functions as a regulator of p53 signaling, apoptosis and autophagy in the antibacterial immune response. Communications Biology, 5, 889.

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Cloning and Characterization of Innate Immune Factors

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The open reading frames (ORFs) of VP35 (KC201176.1), grass carp RIG-I (JX649222.1), MAVS (KF366908.1), TBK1 (JN704345.1), and IRF3 (KT347289.1) were amplified by using a PCR and then cloned into pcDNA3.1(+), pCMV-Myc, pCMV-HA, and pCMV-Tag2C vectors. For the cell location experiment, the ORF of VP35 was inserted into the pEGFP-N3 vector (Clontech, CA, USA). The ORFs of MAVS, TBK1, and IRF3 were also cloned into the pCS2-mCherry vector (Clontech, CA, USA). Insertion of the corresponding 5' flanking regulatory region of an IFN1 promoter (GU139255.1) into a pGL3-Basic luciferase reporter vector (Promega, Madison, WI, USA) to generate an IFN1pro-Luc construct for a promoter activity analysis. The interferon-stimulated response element-(ISRE)-Luc plasmid in pGL3-Basic luciferase reporter vector (Promega, Madison, WI, USA) and DrIFNϕ1pro-Luc were described in a previous study (32 (link)). All constructed expression vectors were verified by DNA sequencing.

Lu L.F., Zhang C., Li Z.C., Zhou X.Y., Jiang J.Y., Chen D., Zhang Y.A, & Li S. (2021). Grass Carp Reovirus VP35 Degrades MAVS Through the Autophagy Pathway to Inhibit Fish Interferon Production. Frontiers in Immunology, 12, 613145.

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Molecular Cloning of Fish Immune Regulators

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The open reading frame (ORF) of GCRV VP56 (KC201172.1) was generated by PCR and then cloned into pcDNA3.1 (+) (Invitrogen), pCMV-Myc (Clontech), or pCMV-HA vectors (Clontech), respectively. The ORFs of grass carp MAVS (KF366908.1), MITA (JN786909.1), TBK1 (JN704345.1), IRF3 (KT347289.1), and IRF7 (KY613780.1) were cloned using the vectors previously described [33 (link)] For subcellular localization, the ORF of VP56 was inserted into pEGFP-N3 vector (Clontech). The ORFs of MAVS, MITA, TBK1, IRF3, and IRF7 were also inserted into pCS2-mCherry vector (Clontech). The expression plasmids for Flag-DrTBK1, HA-DrIRF3, HA-DrIRF7, and Myc- DrIRF7 were described previously [33 (link)]. For promoter activity analysis, IFN1pro-Luc construct was generated by insertion of corresponding 5′-flanking regulatory region of IFN1 promoter (GU139255.1) into pGL3-basic luciferase reporter vector (Promega, Madison, WI). The ISRE-Luc plasmid in the pGL3-basic luciferase reporter vector (Promega) was constructed as described previously [34 (link)]. The Renilla luciferase internal control vector (pRL-TK) was purchased from Promega. The primers including the restriction enzyme cutting sites used for plasmid construction are listed in Supplemental Table I. All constructs were confirmed by DNA sequencing.

Zhang C., Lu L.F., Li Z.C., Zhou X.Y., Zhou Y., Chen D.D., Li S, & Zhang Y.A. (2020). Grass carp reovirus VP56 represses interferon production by degrading phosphorylated IRF7. Fish & Shellfish Immunology, 99, 99-106.

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Mutational Analysis of SNAIL Promoter Activity

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A 2.0-kb SNAIL promoter was cloned into pGL3-Basic Luciferase Reporter Vectors (Promega, USA). Site-specific mutagenesis of the SNAIL promoter was carried out using a QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA), according to the manufacturer's instructions. Primers used to generate the mutant vector were as follows: mut1, 5′-TGTGAGGTTTATGCCAGAGCCACCC-3′ (sense) and 5′-GGGTGGCTCTGGCATAAACCTCACA-3′ (antisense); mut2, 5′-GTAATTATCTGTGCACTTCGTCTGTC-3′ (sense) and 5′-GACAGACGAAGTGCACAGATAATTAC-3′ (antisense); mut1and2, 5′-TGTGAGGTTTATGCCAGAGCCACCC-3′ (sense) and 5′-GACAGACGAAGTGCACAGATAATTAC-3′ (antisense). The mutation was confirmed by DNA sequencing. SNAIL promoter activity was normalized by cotransfection with a ®-actin/Renilla luciferase reporter, containing a full-length Renilla luciferase gene 31 (link). Both, firefly and Renilla luciferase activity were quantified using a Dual-Luciferase Reporter Assay System (Promega, USA) 24 h after transfection.

Wei P., Zhang N., Wang Y., Li D., Wang L., Sun X., Shen C., Yang Y., Zhou X, & Du X. (2015). FOXM1 Promotes Lung Adenocarcinoma Invasion and Metastasis by Upregulating SNAIL. International Journal of Biological Sciences, 11(2), 186-198.

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SIRT5 Promoter Activity Regulation

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The promoter sequence of SIRT5 (chr13: 43,365,384-43,365,730) was obtained from the UCSC Genome Browser (https://genome.ucsc.edu/) and inserted into pGL3-Basic Luciferase Reporter Vectors (Promega Corporation, Madison, WI, USA) to construct the wild type (wt) SIRT5 promoter reporter plasmid. In addition, mutant (mut) SIRT5 promoter reporter plasmids containing mutated SIRT5 promoter sequences were constructed by point mutation. The above plasmids were transfected into cardiomyocytes by Lipofectamin3000 alone (Blank group) or with oe-NC and oe-SPI1, respectively, followed by HG treatment. After 48 h, luciferase activity was detected by the Dual-Luciferase® Reporter Assay System (Promega).

Wei C., Shi M., Dong S., Li Z., Zhao B., Liu D., Li G., Cen J., Yu L., Liang X, & Shi L. (2024). SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy. International Journal of Biological Sciences, 20(2), 585-605.

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Investigating ZFPM2 Promoter Activity

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The promoter of ZFPM2 was inserted into the pGL3 basic luciferase reporter vectors (Promega, Madison WI, USA). ZFPM2 promoter reporter was transfected with pcDNA3.1/ZFPM2‐AS1 or si‐ZFPM2‐AS1#1/2 (with pcDNA3.1 or si‐NC as negative controls) into 293T cells. The luciferase activity of ZFPM2 promoter reporter was normalized to Renilla luciferase reporter. One day after transfection, the detection of luciferase activity was conducted with the application of a Dual‐Luciferase Reporter Assay System (Promega).

Han S., Cao D., Sha J., Zhu X, & Chen D. (2020). LncRNA ZFPM2‐AS1 promotes lung adenocarcinoma progression by interacting with UPF1 to destabilize ZFPM2. Molecular Oncology, 14(5), 1074-1088.

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Luciferase Reporter Assay for SUZ12 and CCDC43 Promoters

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A 1.4-kb SUZ12 or CCDC43 promoter was cloned into pGL3-Basic Luciferase Reporter Vectors (Promega, USA). Empty pGL3-Basic vector was served as a negative control. QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) was applied to generate SUM12-MUT and CCDC43- MUT reporters. The mutation was confirmed by DNA sequencing. The cells were transfected with recombinant plasmids by using Lipofectamine 3000. The pRL-CMV vector (Promega) was served to standardize the transfection efficiency during all transfections. At 36-48 hours after transfection, we collected the cells and incubated with the reporter lysis buffer (Promega). The luciferase activity in cells were tested by a dual luciferase assay kit (Promega) following the instruction of manufacture. Promoter transcription activity was shown as the fold induction of relative luciferase unit (RLU) compared with basic pGL3 vector control. The sequences of oligonucleotide primers applied in this study is outlined in Supplementary Table 1.

Yang Q., Wang Y., Li M., Wang Z., Zhang J., Dai W., Pei M., Hong L., Xiao Y., Hu H., Li J., Lin J., Wu X., Chen Y., Huang M., Li A., Liu S., Tang W., Xiang L, & Wang J. (2021). HMGA1 promotes gastric cancer growth and metastasis by transactivating SUZ12 and CCDC43 expression. Aging (Albany NY), 13(12), 16043-16061.

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DDX5 Promoter Luciferase Assay

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The DDX5 promoter was cloned into the pGL3 basic luciferase reporter vectors (Promega, USA). In total, 5000 cells were seeded into each well of a 96-well plate and transfected with 100 ng of the TOP/FOP-flash reporter plasmids (Millipore, MA, USA), 100 ng of an expression vector (pGL3-DDX5 or pGL3-Basic) or 0.25 μl of siRNA. DDX5 promoter activity and TOP/FOP-flash were normalized by cotransfection with 10 ng of a Renilla luciferase reporter. After 24 h of incubation, the luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega, USA).

Zhang M., Weng W., Zhang Q., Wu Y., Ni S., Tan C., Xu M., Sun H., Liu C., Wei P, & Du X. (2018). The lncRNA NEAT1 activates Wnt/β-catenin signaling and promotes colorectal cancer progression via interacting with DDX5. Journal of Hematology & Oncology, 11, 113.

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Knockdown and Overexpression of UBE2S, FOXM1, and Ub

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Specific shRNAs targeting UBE2S or FOXM1 were cloned into pLKO.1-puro vector (Addgene, USA) for stable knockdown, and the sequences including negative control (NTC) were listed in the supplementary material 1. Full-length coding sequence (CDS) for UBE2S, FOXM1, and Ub protein were cloned into pLenti-CMV-blast vectors (Addgene, USA) for stable overexpression, and the primers for amplification for full-length CDS of these genes were listed in the supplementary material 1. For luciferase assay, wild-type (WT) and mutated (MUT) promoter region ~2000 bp upstream of the transcriptional start site of UBE2S gene were cloned into the pGL3-Basic luciferase reporter vectors (Promega, USA), and the primers were listed in the supplementary material 1.

Gui L., Zhang S., Xu Y., Zhang H., Zhu Y, & Kong L. (2021). UBE2S promotes cell chemoresistance through PTEN-AKT signaling in hepatocellular carcinoma. Cell Death Discovery, 7, 357.

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Luciferase Assay for miR-138-5p Targets

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Luciferase reporter assay was carried out as previously described (6 (link)). Briefly, Lnc-TRPM2-AS, EGFR, or mutant EGFR were predicted as targets of miR-138-5p by RNA22-HAS (https://cm.jefferson.edu/rna22/) and TargetScan V7.2 (http://www.targetscan.org/vert_72/), as shown in Table S1 and Figures S1,S2. The mutant 3'-UTR construct was made by introducing the mismatch mutation into the putative seed regions of EGFR and TRPM2-AS, respectively (Figures S3,S4). Next, the cDNA sequences for TRPM2 and EGFR (GenePharma) were subcloned into pGL3-basic luciferase reporter vectors (Promega, Madison, Wisconsin, USA). NCI-H1299 and A549 cells were seeded into 96-well plates and co-transfected with luc-TRPM2-wt/luc-TRPM2-mut or luc-EGFR-wt/luc-EGFR-mut, and miR138-5p mimic/NC mimic using Lipofectamine 2000 (Invitrogen). At 48 h after transfection, a Dual-Luciferase Reporter Assay System (Promega) was used to measure luciferase activity according to the manufacturer’s instructions. Relative luciferase activity was normalized to Renilla luciferase.

Cui D., Feng Y., Shi K., Zhang H, & Qian R. (2020). Long non-coding RNA TRPM2-AS sponges microRNA-138-5p to activate epidermal growth factor receptor and PI3K/AKT signaling in non-small cell lung cancer. Annals of Translational Medicine, 8(20), 1313.

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