Pgl3 promoter vector

Manufactured by Thermo Fisher Scientific

Sourced in United States, China

The PGL3 promoter vector is a plasmid used in molecular biology experiments. It contains a promoter sequence derived from the Photinus pyralis (firefly) luciferase gene, which can be used to drive the expression of a gene of interest. The vector provides a standardized tool for studying gene regulation and expression in various cell lines and experimental systems.

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PGL3-promoter PGL3 vector

30 protocols using pgl3 promoter vector

Transcription Factor Binding in HOXA11-AS

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Dual-luciferase reporter assays were performed to detect the transcription factors that bind to regions in the HOXA11-AS promoter. Truncated fragments of the HOXA11-AS promoter were amplified and inserted into a pGL3-Basic luciferase reporter (Promega, Madison, WI, USA) by using the restriction enzymes Mlu1 I and XhoI (TaKaRa, Japan); they were then ligated by use of T4 DNA ligase (TaKaRa, Japan) for subsequent transfection into WiT49 cells. Cultured cells that were 80%–90% confluent were transfected using a Lipofectamine 2000 kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). After 48 h of transfection, firefly and Renilla luciferase activities were measured using a Dual Luciferase Reporter Assay System (Promega). Relative luciferase activity was determined by using Renilla luciferase activity as an internal control.
Next, dual-luciferase reporter assays were performed to confirm the binding of HIF-1α, HOXA11-AS, C/EBPβ, and HOXA11-AS. First, the binding sequences for HIF-1α or C/EBPβ on HOXA11-AS were synthesized (WT) and mutated (Mut); after which, pGL3-promoter vectors (Thermo Fisher Scientific) containing the binding sites were constructed by GenePharma (Shanghai, China). Next, the vectors were transfected into WiT49 cells treated with control conditions or hypoxia. Finally, a Dual-Luciferase Reporter Assay System (Promega) was used to detect luciferase activity.

Zhu S., Zhou R., Tang X., Fu W, & Jia W. (2024). Hypoxia/inflammation-induced upregulation of HIF-1α and C/EBPβ promotes nephroblastoma cell EMT by improving HOXA11-AS transcription. Heliyon, 10(6), e27654.

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Validating miR-127-5p and LEF1 Interactions

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The targeting sites of miR-127-5p and CDH11 were predicted by TargetScan and the miRbase database. The wild-type (WT) and mutant (MUT) binding sequences were synthesized by GenePharma (Shanghai, China) then inserted into the pGL3-promoter vectors (Thermo Fisher Scientific). Then, the vectors and miR-127-5p mimics were transfected into SW1353 cells. The Dual-Luciferase Reporter Assay System bought from Promega Corporation (Madison, WI, USA) was employed to detect luciferase activity.
To detect the binding sequence of LEF1 and DNM3OS promoter, the pGL3-promoter vectors of segmented DNM3OS were constructed. To confirm the binding sequence of LEF1 and DNM3OS promoter, dual-fluorescent report gene carrier of DNM3OS WT and MUT sequences was established. Then, the dual-fluorescent report gene carrier were co-transfected into SW1353 with overexpressed LEF1 plasmid and luciferase activity was detected using the above methods.

Wang J., Yang Z., He X., Wang Y., Luo D., Xu W., Zhang H, & Zhou X. (2024). DNM3OS/miR-127-5p/CDH11, activates Wnt3a/β-catenin/LEF-1 pathway to form a positive feedback and aggravate spine facet joint osteoarthritis. Non-coding RNA Research, 9(2), 294-306.

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Cloning and Characterizing CatA Gene Promoter

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The 5′ FLR region of the CatA gene was amplified from human genomic DNA using forward and reverse primers (Forward: 5′-CAGATATGGTACCACGAGGAG-3′, Reverse: 5′-CTGGAAGTGATGTGTACGAGTC-3′). Kpn I (TaKaRa) restriction sites were created in the primers for cloning. The products were cloned into the pGL3 Promoter vector (Promega). K562 cells were plated in a 24-well plate at a density of 5.0 × 10⁴ cells/well and maintained in Opti-MEM with Lipofectamine™2000 (Invitrogen), pRL-TK, and the pGL3 Promoter vectors with 5′ FLR constructs of CatA gene at 37 °C in 5% carbon dioxide. After 24 h, reporter assays were performed using the Dual-Luciferase Reporter Assay System (Promega) and normalized for transfection efficiency by co-transfected Renilla-luciferase.

Ito S., Hirota T., Yanai M., Muto M., Watanabe E., Taya Y, & Ieiri I. (2021). Effects of Genetic Polymorphisms of Cathepsin A on Metabolism of Tenofovir Alafenamide. Genes, 12(12), 2026.

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Dissecting miR-101-3p Regulation of PEG10 and KIF2A

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The 3′UTR fragment of KIF2A (KIF2A-3′UTR-WT) and the wide-type PEG10 sequence (WT-PEG10) containing the predictive binding sites of miR-101-3p were inserted into pGL3 promoter vectors (Invitrogen). The mutant sequences of PEG10 and KIF2A were used to establish PEG10-mutant (MUT-PEG10) and KIF2A-mutant (KIF2A-3′UTR-MUT) reporter vectors. Then, SU-DHL-8 and OCI-LY-8 cells were co-transfected with miR-NC or miR-101-3p mimics and MUT-PEG10, WT-PEG10, KIF2A-3′UTR-WT or KIF2A-3′UTR-MUT. Finally, the dual-luciferase reporter assay system (Promega, Madison, WI, USA) was carried out to determine the luciferase activity in SU-DHL-8 and OCI-LY-8 cells.

Zhao J., Su L, & Jiang J. (2020). Long Non-Coding RNA Paternally Expressed Imprinted Gene 10 (PEG10) Elevates Diffuse Large B-Cell Lymphoma Progression by Regulating Kinesin Family Member 2A (KIF2A) via Targeting MiR-101-3p. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e922810-1-e922810-11.

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Dual-Luciferase Assay for circPVT1 and TRIM23 Regulation

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The wide-type sequences of circPVT1 or TRIM23 3ʹ UTR containing predicted binding sequences of miR-377 and mutant sequences of circPVT1 or TRIM23 3ʹ UTR were constructed and cloned into the pGL3 promoter vectors (Invitrogen) to generate WT-circPVT1, MUT-circPVT1, WT-TRIM23, MUT-TRIM23 luciferase reporter vectors, respectively. Then, the above reporter vectors and miR-377 mimic or miRNA NC were co-transfected into SNU-387 and Huh7 cells. Finally, Dual-Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) was used to detect the relative luciferase activity.

Bu N., Dong Z., Zhang L., Zhu W., Wei F, & Zheng S. (2020). CircPVT1 Regulates Cell Proliferation, Apoptosis and Glycolysis in Hepatocellular Carcinoma via miR-377/TRIM23 Axis. Cancer Management and Research, 12, 12945-12956.

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Luciferase Reporter Assay for CircRNA and mRNA 3'UTR Interactions

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The wide-type sequences of hsa_circ_0002130 (WT-hsa_circ_0002130), GLUT1 (WT-GLUT1 3ʹUTR), HK2 (WT-HK2 3ʹUTR) and LDHA (WT-LDHA 3ʹUTR) were constructed and inserted into pGL3 promoter vectors (Invitrogen). The mutant sequences of hsa_circ_0002130, GLUT1, HK2 and LDHA were used to establish hsa_circ_0002130 mutant (MUT-hsa_circ_0002130), GLUT1 mutant (MUT-GLUT1 3ʹUTR), HK2-mutant (MUT-HK2 3ʹUTR) and LDHA-mutant (MUT-LDHA 3ʹUTR) reporter vectors. HEK293T cells were co-transfected with WT-hsa_circ_0002130, WT-GLUT1 3ʹUTR, WT-HK2 3ʹUTR, WT-LDHA 3ʹUTR or MUT-hsa_circ_0002130, MUT-GLUT1 3ʹUTR, MUT-HK2 3ʹUTR, MUT-LDHA 3ʹUTR reporter vectors and miR-498 mimic or NC mimic. Finally, the relative luciferase activity was detected using a dual-luciferase reporter assay system (Promega, Madison, WI, USA).

Ma J., Qi G, & Li L. (2020). A Novel Serum Exosomes-Based Biomarker hsa_circ_0002130 Facilitates Osimertinib-Resistance in Non-Small Cell Lung Cancer by Sponging miR-498. OncoTargets and therapy, 13, 5293-5307.

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Investigating TMEM16A-miR-381 Interaction

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The wild-type TMEM16A-3′UTR (WT) and mutant TMEM16A-3′UTR (MUT) containing the putative binding site of miR-381 were chemically synthesized and cloned into the downstream of the firefly luciferase gene in a pGL3-promoter vector (Ambion). The gastric cancer cells were seeded in 24-well plates for 24 h, and then were co-transfected with wide-type or mutant-type TMEM16A vector, and agomiR-381 or negative control. After 48 h, cells were harvested, the activities of both firefly and Renilla luciferases in cell lysates were measured using the Dual Luciferase Reporter Assay System (Promega). Renilla luciferase was used for normalization.

Cao Q., Liu F., Ji K., Liu N., He Y., Zhang W, & Wang L. (2017). MicroRNA-381 inhibits the metastasis of gastric cancer by targeting TMEM16A expression. Journal of Experimental & Clinical Cancer Research : CR, 36, 29.

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Luciferase Assay for FOXM1 3'-UTR

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The wild-type (wt) 3′-UTR of FOXM1 or mutant type (mut) 3′-UTR of FOXM1 containing the binding site of miR-215-3p was constructed and inserted into the downstream of the firefly luciferase gene in a pGL3-promoter vector (Ambion); HEK-293T cell (1 × 10⁵) was cultured in 96-well plates for 24 hours and cells were transfected with wt or mut 3′-UTR of FOXM1 and miR-NC or miR-215-3p using Lipofectamine 2000 reagent (Thermo Fisher Scientific). Cell lysates were collected 24 hours after transfection, then luciferase activity was detected using a Luciferase Reporter System (Promega).

Tang X., Shi X., Wang N., Peng W, & Cheng Z. (2019). MicroRNA-215-3p Suppresses the Growth, Migration, and Invasion of Colorectal Cancer by Targeting FOXM1. Technology in Cancer Research & Treatment, 18, 1533033819874776.

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Validating miR-22-3p Target on VE-cadherin

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To examine whether miR-22-3p targets VE-cadherin directly, we constructed wild-type CDH5 3′UTR reporter plasmid (CDH5 WT) and mutated-type CDH5 3′UTR reporter plasmid (CDH5 MUT) with pGL3-promoter vector (Ambion). 293T cells were placed on 24-well plates and cultured in DMEM with 10% fetal bovine serum (FBS) at 37°C in an incubator containing 5% CO₂ until it has grown to 60% confluence. And then, 293T cells were cotransfected with luciferase plasmids and miR-22-3p mimic or negative control (NC). After 48h transfection, luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.

Zhang W., Zhang T., Yan Y., Zhang J., Zhou Y., Pei Y., Yao L., You B, & Chen J. (2020). Exosomal miR-22-3p Derived from Chronic Rhinosinusitis with Nasal Polyps Regulates Vascular Permeability by Targeting VE-Cadherin. BioMed Research International, 2020, 1237678.

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Luciferase Assay for miRNA Binding

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The wild-type PTEN-3ʹ-UTR was chemically synthesized and cloned downstream of the firefly luciferase gene in a pGL3-promoter vector (Ambion). 293-T cells were placed in a 48-well plate and grown until 80% confluence. Cells were then cotransfected with luciferase plasmids, pRL-TK Renilla plasmid and miR-301a or miR-301a-mut. After 48 h of transfection, firefly and Renilla luciferase activities were evaluated with a Dual-Luciferase Reporter Assay System (Promega).

Li J., Jiang D., Zhang Q., Peng S., Liao G., Yang X., Tang J., Xiong H, & Pang J. (2020). MiR-301a Promotes Cell Proliferation by Repressing PTEN in Renal Cell Carcinoma. Cancer Management and Research, 12, 4309-4320.

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