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Prl sv40

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The PRL-SV40 is a plasmid vector used in molecular biology research. It contains the SV40 large T antigen gene, which can be used to immortalize certain cell lines. The core function of this product is to enable the long-term proliferation of specific cell types in culture.

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Lab products found in correlation

Lenticrisprv2 puro, by Addgene (2 mentions) Tripz shrna plasmids, by Horizon Discovery (2 mentions) Pgl3 ap 1, by Addgene (2 mentions) Pspax2, by Addgene (2 mentions) Pgl4.29 luc2p cre hygro, by Promega (1 mentions) Pgl4.33 luc2p sre hygro, by Promega (1 mentions) 4xm67 ptata tk luc, by Addgene (1 mentions) M50 super 8x topflash, by Addgene (1 mentions) M51 super 8x fopflash topflash mutant, by Addgene (1 mentions) Dual luciferase reporter assay kit, by Promega (1 mentions) Transit lt1 transfection reagent, by Mirus Bio (1 mentions) Lithium chloride (licl), by Merck Group (1 mentions) Pspcas9 bb 2a gfp px458, by Addgene (1 mentions) α rabbit hrp, by Jackson ImmunoResearch (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by R&D Systems (1 mentions)

6 protocols using prl sv40

1

Zebrafish Genetic Manipulation Protocol

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An sgRNA plasmid (MITF GFP 2X-sgRNA), which harbors a mitfa:GFP insert as well as a 2X sgRNA cassette (Fig. 2A), was generated for the zebrafish screen. The 2X sgRNA cassette from MAZERATI 2X (MAS2X) sgRNA (RRID Addgene_118844), also called MiniCoopR 2X sgRNA (gift from Dr. Len Zon), was amplified and introduced into mitfa:GFP plasmid using In-fusion cloning. Site-directed mutagenesis was performed to introduce NheI sites spanning sgRNA2 of the plasmid. MAS2X sgpten was cloned from MAS2X sgRNA (Supplementary Fig. S2A; ref. 44, 109 (link)). The U6-sgRNA plasmid (RRID Addgene_64245) was used along with mitfa:Cas9 plasmid and MiniCoopR:tdTomato (previously developed in the lab). LentiCRISPRV2 Puro (RRID Addgene_52961), psPAX2 (RRID Addgene_12260), and MD2 (RRID Addgene_12259) plasmids were obtained from Addgene. TRIPZ shRNA plasmids were purchased from Dharmacon expressing a nontargeting (NT) shRNA or shRNAs against human GRAMD1B. Clone IDs and hairpin sequences are in Supplementary Table S3. Control luciferase plasmid (pGL3, RRID Addgene_48743) and Renilla luciferase plasmid (pRL-SV40, RRID Addgene_27163) were obtained from Promega (E1751 and E2231) and pGL3-AP-1 was from Addgene (RRID Addgene_40342). Details of the oligonucleotide sequences used for cloning and sequencing are in Supplementary Table S3.
Suresh S., Rabbie R., Garg M., Lumaquin D., Huang T.H., Montal E., Ma Y., Cruz N.M., Tang X., Nsengimana J., Newton-Bishop J., Hunter M.V., Zhu Y., Chen K., de Stanchina E., Adams D.J, & White R.M. (2022). Identifying the Transcriptional Drivers of Metastasis Embedded within Localized Melanoma. Cancer Discovery, 13(1), 194-215.
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2

Plasmid Transfection Assay Protocol

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CISH reporter plasmid was kindly supplied by Dr. Clevenger [21 (link)]. pGL4.33[luc2P/SRE/Hygro] (MEK-Erk response element, Promega #E1340); pGL4.29[luc2P/CRE/Hygro] (cAMP response element, Promega #E8471); 4xM67 pTATA TK-Luc (Stat3 response element, Addgene #8688) are commercially available. pRL-SV40 (Addgene #E2231) was used as transfection efficiency control.
Cuesta-Casanovas L., Delgado-Martínez J., Cornet-Masana J.M., Carbó J.M., Banús-Mulet A., Guijarro F., Esteve J, & Risueño R.M. (2023). Prolactin receptor signaling induces acquisition of chemoresistance and reduces clonogenicity in acute myeloid leukemia. Cancer Cell International, 23, 97.
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3

Evaluating Wnt Signaling Modulators

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A549-AT cells were seeded in a 96-well format and were transfected with 0.1 ng of pRL-SV40 (Addgene; #27163) together with either 0.1 μg M50 Super 8x TOPFlash (Addgene; #12456) or M51 Super 8x FOPFlash (TOPFlash mutant; Addgene; #12457) using TransIT®-LT1 Transfection Reagent (Mirus Bio LLC; Madison, Wisconsin, United States). After 24 h, cells were treated with either of the following substances: 10 mM LiCl (Merck KgaA; Darmstadt, Germany), 100 μg/mL rhWnt-3a, 10/3/1/0.3 μM PRI-724. Additionally, cells were treated simultaneously with LiCl and PRI-724. After 24 h, cells were analyzed for luciferase activity using a Dual Luciferase Reporter Assay Kit (Promega; Madison, Wisconsin, United States) according to manufacturer’s instructions.
Kelch M.A., Vera-Guapi A., Beder T., Oswald M., Hiemisch A., Beil N., Wajda P., Ciesek S., Erfle H., Toptan T, & Koenig R. (2023). Machine learning on large scale perturbation screens for SARS-CoV-2 host factors identifies β-catenin/CBP inhibitor PRI-724 as a potent antiviral. Frontiers in Microbiology, 14, 1193320.
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4

Investigating STK11 and p53 Signaling

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Dulbecco’s modified eagle’s medium and RPMI 1640 were purchased from Thermo Scientific (Waltham, MA), fetal bovine serum was obtained from R&D Systems (Minneapolis, MN). Wild-type (WT; Addgene #8590) and kinase-dead (Addgene #8591) STK11 plasmids along with the p53-driven firefly luciferase (PG13-luc) and constitutive Renilla luciferase (pRL-SV40) plasmids, and the Cas9 and sgRNA plasmid used for asymmetric repair (pSpCas9(BB)-2A-GFP (PX458)) were obtained from Addgene (Cambridge, MA). Antibodies directed against LKB1 (E-9; #sc-374334), p53 (D0-1; #sc-126), Actin (C-2; #sc-8432), STRADα (G-8; sc 515635), MO25 (#2716) and Tubulin (#2144) were obtained from Santa Cruz Biotechnology (Dallas, TX) and Cell Signaling Technology (Danvers, MA), respectively. Secondary antibodies (α-mouse-horseradish peroxidase [HRP] and α-rabbit-HRP) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All other reagents were purchased from Fisher Scientific (Waltham, MA) unless otherwise noted.
Donnelly L.L., Hogan T.C., Lenahan S.M., Nandagopal G., Eaton J.G., Lebeau M.A., McCann C.L., Sarausky H.M., Hampel K.J., Armstrong J.D., Cameron M.P., Sidiropoulos N., Deming P, & Seward D.J. (2021). Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers. Carcinogenesis, 42(12), 1428-1438.
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5

Irak-1 3'UTR Luciferase Assay

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HEK293 cells in 96-well plates were co-transfected with 100 ng of CMV-luciferase-Irak-1 3′UTR vector (Addgene, Watertown, MA), 5 pmol of ds or ss miR-146a mimics and 10 ng of pRL-SV40 (Addgene, Watertown, MA) for normalization of transfection efficiency. Luciferase activities were measured 48 hours later.
Wang S., Yang Y., Suen A., Zhu J., Williams B., Hu J., Chen F., Kozar R., Shen S., Li Z., Jeyaram A., Jay S.M., Zou L, & Chao W. (2021). Role of extracellular microRNA-146a-5p in host innate immunity and bacterial sepsis. iScience, 24(12), 103441.
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6

Generation of a MITF-GFP Zebrafish Screening Plasmid

Cited 1 time
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A sgRNA plasmid (MITF GFP 2X-sgRNA) which harbors a mitfa:GFP insert as well as a 2X sgRNA cassette (Fig.2A) was generated for the zebrafish screen. The 2X sgRNA cassette from MAZERATI 2X (MAS2X) sgRNA (RRID Addgene_118844), also called MiniCoopR 2X sgRNA, gift from Dr. Len Zon) was amplified and introduced into mitfa: GFP plasmid using In-fusion cloning. Site directed mutagenesis was performed to introduce NheI sites spanning sgRNA2 of the plasmid. MAS2X sgpten was cloned from MAS2X sgRNA (Supp. Fig. 2A) (44 (link),109 (link)). The U6-sgRNA plasmid (RRID Addgene_64245) was used along with mitfa:Cas9 plasmid and MiniCoopR:tdTomato (previously developed in the lab). LentiCRISPRV2 Puro (RRID Addgene_52961), psPAX2 (RRID Addgene_12260) and MD2 (RRID Addgene_12259) plasmids were obtained from Addgene. TRIPZ shRNA plasmids were purchased from Dharmacon expressing a non-targeting shRNA or shRNAs against human GRAMD1B. cloneIDs and hairpin sequences are in Supp. Table. S3. Control luciferase plasmid (pGL3 RRID Addgene_48743 ) and Renilla luciferase plasmid (pRL-SV40, RRID Addgene_27163) was obtained from Promega (E1751 and E2231) and pGL3-AP-1 was from Addgene (RRID Addgene_40342). Details of the oligonucleotide sequences used for cloning and sequencing are in Supp. Table. S3.
Suresh S., Rabbie R., Garg M., Lumaquin D., Huang T.H., Montal E., Ma Y., Cruz N.M., Tang X., Nsengimana J., Newton-Bishop J., Hunter M.V., Zhu Y., Chen K., de Stanchina E., Adams D.J, & White R.M. (2022). Identifying the Transcriptional Drivers of Metastasis Embedded within Localized Melanoma. Cancer Discovery, 13(1), 194-215.
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