Pgl3 promoter

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PGL3-promoter is a laboratory equipment component used in gene expression studies. It serves as a promoter sequence that can drive the expression of reporter genes, enabling researchers to investigate and measure gene regulation mechanisms. The core function of the PGL3-promoter is to facilitate the transcription of the associated reporter gene, providing a tool for researchers to study various aspects of gene expression in their experimental systems.

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Lab products found in correlation

Smai restriction enzyme, by Takara Bio (1 mentions) Passive lysis buffer (plb), by Promega (1 mentions) Luciferase reporter assay kit, by Promega (1 mentions) Prl cmv, by Promega (1 mentions) Pgl3 promoter vector, by Thermo Fisher Scientific (1 mentions) Dual glo luciferase assay, by Promega (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Pgl3 promoter, by Promega (1 mentions) Lipofectamine 3000 kit, by Thermo Fisher Scientific (1 mentions) Dual luciferase reporter assay system, by Promega (1 mentions)

Close spelling variants of this product

PGL3 promoter vector

4 protocols using pgl3 promoter

Zfp819 Transcriptional Regulation Assay

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GC-2 cells (4.0 × 10⁵ cells/well) were seeded onto 24-well plates and incubated at 37 °C for 24 h. For repressive activity of Zfp819-KRAB, when the cells reached approximately 80–90% confluence, they were co-transfected with 250 ng of pcDNA3.1/myc-Zfp819-KRAB, 250 ng of a firefly luciferase-encoding vector (pGL3-promoter, Invitrogen), and 5 ng of pRL-TK (Renilla used as an internal control for normalization of transfection efficiency in the absence and presence of Zfp819 overexpression). After 24 h, the cells were lysed with passive lysis buffer (Promega), dual luciferase assays were performed with a Luciferase Reporter Assay kit (Promega), and the luciferase activity was measured using a Centro LB 960 DLReady microplate illuminometer (Berthold Technologies). GAL4-DBD was used as a basic control, and KOX1-DBD was used as a positive control. For promoter activity of Zfp819, Tnrc6b or Anxa11 promoter regions were inserted into pGL-promoter (Invitrogen). When the cells reached approximately 80–90% confluence, they were co-transfected with 250 ng of pcDNA3.1/myc-Zfp819 or pcDNA3.1/myc, 250 ng of a firefly luciferase-encoding vector (pGL3-promoter, Invitrogen), and 5 ng of pRL-TK (Renilla). Each experiment was repeated three independent times in triplicate.

Jin S., Choi H., Kwon J.T., Kim J., Jeong J., Kim J., Hong S.H, & Cho C. (2017). Identification of target genes for spermatogenic cell-specific KRAB transcription factor ZFP819 in a male germ cell line. Cell & Bioscience, 7, 4.

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Plasmid Constructs for p53 and DNA Structures

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Plasmids encoding human p53 proteins pT7-7wtp53 (full length wild type p53,p53, aa 1–393), pET-p53CD (p53CD, aa 94–312), pGEX-2TKp53CT (GST-p53CT, aa 320–393), pGEX-2TKp53T (GST-p53T, aa 363–393) and pGEX-4Tp53CD (GST-p53CD, aa 94–312) were described in [10 (link), 29 (link)]. Plasmids with T.A.T triplex forming sequences (pBA50 and pPA50) were prepared by cloning of (dT)₅₀.(dA)₅₀ into the EcoRV site of pBluescript SK II- (pBSK, Stratagene) and pPGM1 [34 (link)] (S1 Table). Similarly, plasmids for cruciform formation (pBAT34, pPAT34) were prepared by cloning (dAdT)₃₄ sequences to the same plasmids, for details see S1 Table. Plasmid pA69 with (dT)₆₉.(dA)₆₉ (on pUC19 basis [35 (link)]) and pUC19 control plasmid were used. Nonspecific competitor (pBSK/SmaI) was prepared by SmaI restriction enzyme (Takara, Japan) cleavage of pBSK. Plasmids for luciferase reporter assay (pGL3-BSK, pGL3-P1, pGL3-BA50, pGL3-PA50, pGL3-PA20_,S1 Table) were constructed by cloning fragments from pBSK derivatives into the SmaI/XhoI site of the pGL3-promoter (Invitrogen). All plasmids were isolated from bacterial strain TOP10 (Stratagene) and verified by sequencing.

Brázdová M., Tichý V., Helma R., Bažantová P., Polášková A., Krejčí A., Petr M., Navrátilová L., Tichá O., Nejedlý K., Bennink M.L., Subramaniam V., Bábková Z., Martínek T., Lexa M, & Adámik M. (2016). p53 Specifically Binds Triplex DNA In Vitro and in Cells. PLoS ONE, 11(12), e0167439.

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Allelic Enhancer Activity Profiling

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Genomic tiles spanning regions containing SNPs with indication of regulatory activity by RegulomeDB were generated. Regions containing the major and minor alleles within the 2p23.2 region spanning 2,229 bp (chr2:29,117,333-29,119,561) were generated by PCR using BAC DNA CTD-3216P10 as template. Forward and reverse primers contained attB1 and attB2 sequences, respectively, to aid in recombinational cloning. Tiles were cloned in both a forward and reverse orientation upstream of the SV40 promoter by recombination in the firefly luciferase reporter vector pGL3-Pro-attb vector designed to test for enhancer regions. This vector is a modification of pGL3-Promoter (Invitrogen) adding attB sites surrounding the ccdb gene. The clone containing the tile was co-transfected in eight replicates using LipoFectamine 2000 (Life Technologies) into MCF10A or CAL51 cells with pRL-CMV (Promega), an internal control expressing Renilla luciferase, per well of 96-well plates. Luciferase activity was measured 24-h post transfection by Dual Glo Luciferase Assay (Promega). Transfections were repeated in two independent experiments with similar results. The influence of the common and rare alleles of rs4407214 on promoter activity in the pGL3-Promoter vector (Invitrogen) were assessed using the same methodology. Primers are available on request.

Couch F.J., Kuchenbaecker K.B., Michailidou K., Mendoza-Fandino G.A., Nord S., Lilyquist J., Olswold C., Hallberg E., Agata S., Ahsan H., Aittomäki K., Ambrosone C., Andrulis I.L., Anton-Culver H., Arndt V., Arun B.K., Arver B., Barile M., Barkardottir R.B., Barrowdale D., Beckmann L., Beckmann M.W., Benitez J., Blank S.V., Blomqvist C., Bogdanova N.V., Bojesen S.E., Bolla M.K., Bonanni B., Brauch H., Brenner H., Burwinkel B., Buys S.S., Caldes T., Caligo M.A., Canzian F., Carpenter J., Chang-Claude J., Chanock S.J., Chung W.K., Claes K.B., Cox A., Cross S.S., Cunningham J.M., Czene K., Daly M.B., Damiola F., Darabi H., de la Hoya M., Devilee P., Diez O., Ding Y.C., Dolcetti R., Domchek S.M., Dorfling C.M., dos-Santos-Silva I., Dumont M., Dunning A.M., Eccles D.M., Ehrencrona H., Ekici A.B., Eliassen H., Ellis S., Fasching P.A., Figueroa J., Flesch-Janys D., Försti A., Fostira F., Foulkes W.D., Friebel T., Friedman E., Frost D., Gabrielson M., Gammon M.D., Ganz P.A., Gapstur S.M., Garber J., Gaudet M.M., Gayther S.A., Gerdes A.M., Ghoussaini M., Giles G.G., Glendon G., Godwin A.K., Goldberg M.S., Goldgar D.E., González-Neira A., Greene M.H., Gronwald J., Guénel P., Gunter M., Haeberle L., Haiman C.A., Hamann U., Hansen T.V., Hart S., Healey S., Heikkinen T., Henderson B.E., Herzog J., Hogervorst F.B., Hollestelle A., Hooning M.J., Hoover R.N., Hopper J.L., Humphreys K., Hunter D.J., Huzarski T., Imyanitov E.N., Isaacs C., Jakubowska A., James P., Janavicius R., Jensen U.B., John E.M., Jones M., Kabisch M., Kar S., Karlan B.Y., Khan S., Khaw K.T., Kibriya M.G., Knight J.A., Ko Y.D., Konstantopoulou I., Kosma V.M., Kristensen V., Kwong A., Laitman Y., Lambrechts D., Lazaro C., Lee E., Le Marchand L., Lester J., Lindblom A., Lindor N., Lindstrom S., Liu J., Long J., Lubinski J., Mai P.L., Makalic E., Malone K.E., Mannermaa A., Manoukian S., Margolin S., Marme F., Martens J.W., McGuffog L., Meindl A., Miller A., Milne R.L., Miron P., Montagna M., Mazoyer S., Mulligan A.M., Muranen T.A., Nathanson K.L., Neuhausen S.L., Nevanlinna H., Nordestgaard B.G., Nussbaum R.L., Offit K., Olah E., Olopade O.I., Olson J.E., Osorio A., Park S.K., Peeters P.H., Peissel B., Peterlongo P., Peto J., Phelan C.M., Pilarski R., Poppe B., Pylkäs K., Radice P., Rahman N., Rantala J., Rappaport C., Rennert G., Richardson A., Robson M., Romieu I., Rudolph A., Rutgers E.J., Sanchez M.J., Santella R.M., Sawyer E.J., Schmidt D.F., Schmidt M.K., Schmutzler R.K., Schumacher F., Scott R., Senter L., Sharma P., Simard J., Singer C.F., Sinilnikova O.M., Soucy P., Southey M., Steinemann D., Stenmark-Askmalm M., Stoppa-Lyonnet D., Swerdlow A., Szabo C.I., Tamimi R., Tapper W., Teixeira M.R., Teo S.H., Terry M.B., Thomassen M., Thompson D., Tihomirova L., Toland A.E., Tollenaar R.A., Tomlinson I., Truong T., Tsimiklis H., Teulé A., Tumino R., Tung N., Turnbull C., Ursin G., van Deurzen C.H., van Rensburg E.J., Varon-Mateeva R., Wang Z., Wang-Gohrke S., Weiderpass E., Weitzel J.N., Whittemore A., Wildiers H., Winqvist R., Yang X.R., Yannoukakos D., Yao S., Zamora M.P., Zheng W., Hall P., Kraft P., Vachon C., Slager S., Chenevix-Trench G., Pharoah P.D., Monteiro A.A., García-Closas M., Easton D.F, & Antoniou A.C. (2016). Identification of four novel susceptibility loci for oestrogen receptor negative breast cancer. Nature Communications, 7, 11375.

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Functional Validation of EDNRB Intronic SNPs

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The 25 SNPs found in the 2nd intron region of EDNRB were located in the functional regulatory region. In order to verify its function, we conducted dual-luciferase expression assays. To generate luciferase reporter constructs for the 25-SNPs haplotype of the EDNRB gene, we cloned the fragments of the Jianli and Xidu black EDNRB gene, respectively, into the pGL3-promoter (Promega, USA) vector. Then transfect the pGL3-promoter vectors and pRL-TK vector into PK-15 cells by Lipofectamine 3000 kit (Invitrogen, Carlsbad, CA, USA). Following the protocol of a dual-luciferase reporter assay system (Promega, Madison, WI, Germany) to quantify luciferase activity, it was calculated as the firefly fluorescence value / renilla fluorescence value (n = 3). Statistical analysis was conducted using a Student’s t-test, and a p-value lower than 0.05 was considered significant.

Xu Z., Wu J., Zhang Y., Qiao M., Zhou J., Feng Y., Li Z., Sun H., Lin R., Song Z., Zhao H., Li L., Chen N., Li Y., Oyelami F.O., Peng X, & Mei S. (2024). Genome-wide detection of selection signatures in Jianli pigs reveals novel cis-regulatory haplotype in EDNRB associated with two-end black coat color. BMC Genomics, 25, 23.

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