Pgl3 basic reporter plasmid

Manufactured by Promega

Sourced in United States

The PGL3-basic reporter plasmid is a DNA vector used in molecular biology experiments. It contains a promoterless luciferase gene, which can be used to measure gene expression levels when inserted into a specific DNA sequence of interest.

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Lab products found in correlation

Prl tk, by Promega (2 mentions) Dual luciferase reporter assay system, by Promega (2 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Pnfκb luc, by Beyotime (1 mentions) Brefeldin a, by Merck Group (1 mentions) Glomax multi detection system, by Promega (1 mentions) Prl sv40, by Promega (1 mentions) Dual luciferase assay kit, by Beyotime (1 mentions) Tca8113, by Procell (1 mentions) Cal 27, by Procell (1 mentions) Z1 inverted microscope, by Zeiss (1 mentions) Prl tk control plasmid, by Promega (1 mentions) Cytoflex s, by Beckman Coulter (1 mentions) Fluostar omega microplate reader, by BMG Labtech (1 mentions) Bx53 upright microscope, by Olympus (1 mentions)

Spelling variants of this product

PGL3-Basic (590 citations) PGL3-basic plasmid (138 citations) PGL3 plasmid (94 citations) PGL3-basic luciferase reporter plasmid (40 citations) PGL3 reporter plasmid (13 citations) PGL3 basic luciferase reporter plasmid vector (4 citations) Reporter plasmids (3 citations) PGL3-basic firefly luciferase reporter plasmid (3 citations)

7 protocols using pgl3 basic reporter plasmid

Pediatric Glioma Cell Lines and Molecular Manipulations

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Pediatric glioma cell lines used in this study were kindly provided by Pediatric Oncology, the Institute of Cancer Research, Sutton, United Kingdom [53 (link)], and included Res259 (pediatric astrocytoma, Grade II), UW479 (pediatric anaplastic astrocytoma, Grade III) and SF188 (pediatric glioblastoma multiforme, Grade IV). Glioma cells and embryonic kidney 293T cells were cultured in Dulbecco's Modified Eagle Medium-F12 (DMEM-F12) and Dulbecco's Modified Eagle Medium (DMEM)(GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO) in 5% CO₂ at 37°C, respectively.
shCENPE, shKIF14, shNCAPG and a control plasmid were obtained from the RNAi consortium at Academia Sinica, Taiwan. For miRNA expression using a plasmid, human miR-137 and miR-6500-3p were PCR-amplified from genomic DNA (Table S3) and cloned into the pLenti4 expression vector (Invitrogen). miRZip-137, which allows for stable expression of anti-microRNA-137(a lentivirus-based plasmid), was purchased from System Biosciences (SBI). All 3′UTR reporter plasmids were PCR-amplified from human genomic DNA (Table S3) and cloned into the pGL3-Basic reporter plasmid (Promega).

Liang M.L., Hsieh T.H., Ng K.H., Tsai Y.N., Tsai C.F., Chao M.E., Liu D.J., Chu S.S., Chen W., Liu Y.R., Liu R.S., Lin S.C., Ho D.M., Wong T.T., Yang M.H, & Wang H.W. (2016). Downregulation of miR-137 and miR-6500-3p promotes cell proliferation in pediatric high-grade gliomas. Oncotarget, 7(15), 19723-19737.

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Evaluating NF-κB Pathway Regulation

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Ectopic MT2A, short hairpin RNA against MT2A (shMT2A-1 and −2), and luciferase reporter containing IκB-α promoter region (pIκB-α-Luc) were constructed and transfected into BGC823 cells for transient expression as described (3 (link), 44 (link)). Luciferase reporter plasmids, pIκB-α-4-Luc and pIκB-α-5-Luc, were constructed by cloning the proximal promoter sequence of the IκB-α gene luciferase reporter construct into BglII/HindIII sites of the pGL3-Basic reporter plasmid (Promega). NF-κB-luciferase reporter plasmid (pNF-κB-Luc) was purchased from Beyotime, Ltd. (D2206). The internal control vector, pRL-TK, was purchased from Promega. In vitro transfection was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. All oligonucleotide sequences are listed in Supplementary Table S1.

Pan Y., Lin S., Xing R., Zhu M., Lin B., Cui J., Li W., Gao J., Shen L., Zhao Y., Guo M., Wang J.M., Huang J, & Lu Y. (2016). Epigenetic Upregulation of Metallothionein 2A by Diallyl Trisulfide Enhances Chemosensitivity of Human Gastric Cancer Cells to Docetaxel Through Attenuating NF-κB Activation. Antioxidants & Redox Signaling, 24(15), 839-854.

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Proximal Ppargc1a Promoter Cloning

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A 1.8 kb region of the proximal Ppargc1a promoter was cloned from HK2 cells into the pGL3-basic reporter plasmid (Promega). TK10 cells were transiently transfected with this construct along with CMV-Renilla reporter to monitor transfection efficiency. Firefly and renilla luciferase levels were quantified with Bright-Glo and Renilla luciferase assays (Promega).

LaGory E.L., Wu C., Taniguchi C.M., Ding C.K., Chi J.T., von Eyben R., Scott D.A., Richardson A.D, & Giaccia A.J. (2015). Suppression of PGC-1α is critical for reprogramming oxidative metabolism in renal cell carcinoma. Cell reports, 12(1), 116-127.

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Promoter-driven Luciferase Assay

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MFM223 and MDAMB453 cells were plated at 5 × 10⁴ cells/dish and cultured for 2 days. Cells were transfected with 0.2 μg of the pGL3 basic reporter plasmid (Promega) carrying the firefly luciferase gene (SKP2-Luc) driven by the human Skp2 promoter or mutant reporter constructs harboring a Skp2 promoter mutant lacking the CRE-binding site (ΔCRE-Luc) or the Skp2 promoter in which the CRE-binding site was mutated (mut-CRE-Luc) together with 0.02 μg of the pRL-SV40 co-reporter plasmid carrying the Renilla luciferase gene (Promega). Transfections were performed using Avalanche-Everyday Transfection Reagent (EZ Biosystems). The Skp2 promoter was identified using the University of California, Santa Cruz genome browser (http://genome.ucsc.edu/cgi-bin/hgGateway; ref. 40 (link)). SKP2-Luc reporter constructs were generated by PCR; the ΔCRE-Luc and mut-CRE-Luc constructs were generated by inverse PCR using SKP2-Luc as a template.
To enhance cleavage of AIbZIP, cells were treated with 1 μmol/L brefeldin A (Sigma-Aldrich) for 24 hours. After 36 hours, luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega) and a GloMax Multi+ Detection System (Promega) following the manufacturer's protocol. Relative luciferase activities were determined as the ratio between firefly and Renilla luciferase activities.

Ito T., Saito A., Kamikawa Y., Nakazawa N, & Imaizumi K. (2024). AIbZIP/CREB3L4 Promotes Cell Proliferation via the SKP2-p27 Axis in Luminal Androgen Receptor Subtype Triple-Negative Breast Cancer. Molecular Cancer Research, 22(4), 373-385.

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Promoter Variants Regulate NFKB1 Expression

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About −800 to 60 region of the NFKB1 promoter containing −94 Ins allele (rs28362491) and −449 C allele (rs72696119) were synthesized and constructed into the pGL3‐Basic reporter plasmid (Promega, Madison, WI) named PGL3‐NFKB1‐IC. Using site‐directed mutagenesis technique, PGL3‐NFKB1‐IC was then mutated to PGL3‐NFKB1‐DC, PGL3‐NFKB1‐IG, and PGL3‐NFKB1‐DG, respectively. Sequence analysis was performed to confirm these plasmids. Subsequently, human tongue squamous cell carcinoma cell lines Tca‐8113 (Procell, Wuhan, China) and CAL‐27 (Procell) in 12‐well plates were transfected with reporter plasmids (1.0 μg) and plasmid pRL‐TK (1.0 μg) (Promega) as an internal control containing renilla luciferase reporter gene. After 12 h, luciferase activities were detected using a dual‐luciferase assay kit (Beyotime, Beijing, China) following the manufacturer's instructions. Relative luciferase activity was calculated according to the RLU (relative light unit) of the firefly luciferase divided by the RLU of the renilla luciferase.

Chen F., Liu F., Yan L., Lin L., Qiu Y., Wang J., Wu J., Bao X., Hu Z., Cai L, & He B. (2018). A functional haplotype of NFKB1 influence susceptibility to oral cancer: a population‐based and in vitro study. Cancer Medicine, 7(5), 2211-2218.

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Nfkbia Promoter Luciferase Assay

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Assays were performed with pGL3-basic reporter plasmid (Promega, Madison, USA) and pRL-TK control plasmid (Promega). Nfkbia promoter region (-977 ~ +34, NM_010907), a truncated promoter region and a mutant TEAD4-binding site fragment were cloned into the multiple cloning site. To visualize Nfkbia-driven activity, red fluorescent protein (RFP) was cloned into the multiple cloning site (pRFP-Nfkbia). Cells of 80% confluence were transfected with reporter plasmids and/or overexpression plasmids as indicated. The Dual-Luciferase^® Reporter Assay System (Promega) was used to detect Firefly (control signal) and Renilla luciferase activities with a FLUOstar Omega microplate reader (BMG LABTECH, Ortenberg, Germany) 48h after transfection. The shRNA-GFP-positive Hepa1-6 clones were transfected with pRFP-Nfkbia for an additional 48h in chamber slides. The slides were fixed with 4% PFA and RFP signals were visualized with a BX53 upright Microscope (Olympus) or an AxioObserver. Z1 inverted microscope (ZEISS). RFP-positive cells were counted with a CytoFLEX S (Beckman, Brea, USA).

Luo X., Zhang R., Lu M., Liu S., Baba H.A., Gerken G., Wedemeyer H, & Broering R. (2021). Hippo Pathway Counter-Regulates Innate Immunity in Hepatitis B Virus Infection. Frontiers in Immunology, 12, 684424.

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Cloning and Mutagenesis of KCNQ1OT1 Promoter

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A 2462 bp DNA fragment encompassing the MAP3K1 transcription start site and a 568 bp KCNQ1OT1 promoter fragment were respectively amplified by PCR using genomic DNA from NB4 cells. The PCR products were cloned into the pGL3-basic reporter plasmid (Promega, Madison, WI, USA). Mutations of the predicted c-Myc binding sites in the pGL3-KCNQ1OT1 construct were made using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s protocol. The c-Myc sequence was amplified using NB4 cDNA and then cloned into the pcDNA3.1 (+) vector. Detailed primer information is listed in Table S2.

Tang D., Luo Y., Jiang Y., Hu P., Peng H., Wu S., Zhang G, & Wang Y. (2021). LncRNA KCNQ1OT1 activated by c-Myc promotes cell proliferation via interacting with FUS to stabilize MAP3K1 in acute promyelocytic leukemia. Cell Death & Disease, 12(9), 795.

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