Plvx ires mcherry vector

Manufactured by Takara Bio

Sourced in United States

The PLVX-IRES-mCherry vector is a plasmid that contains an internal ribosome entry site (IRES) sequence and the mCherry fluorescent protein. This vector can be used to express two genes from a single promoter, with the mCherry protein serving as a reporter for gene expression.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions) Secrete pair gaussia luciferase assay kit, by GeneCopoeia (1 mentions) Synergy 2 luminescence plate reader, by Agilent Technologies (1 mentions) Psicor vector, by Addgene (1 mentions) Gipz lentiviral shrna vector, by Horizon Discovery (1 mentions) Plvx ires puro vector, by Takara Bio (1 mentions) Trans lentiviral shrna packaging kit with calcium phosphate transfection reagent, by Horizon Discovery (1 mentions)

Close spelling variants of this product

PLVX vector (22 citations) PLVX-IRES-mCherry (16 citations) PLVX-EF1alpha-IRES-mCherry Vector (6 citations) PLVX-EF1a-IRES- mCherry Vector (2 citations)

4 protocols using plvx ires mcherry vector

Generation of TRPV4 Constructs for Research

Cited 2 times

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The constructs TRPV4^FeRIC, TRPV4^ΔTFeRIC, and TRPV4^WT were obtained as described previously (Hutson et al., 2017 (link); Hernández-Morales et al., 2020 (link)). To generate the TRPV4^WT construct, we used full-length rat TRPV4 cDNA, which was a gift from R. Lefkowitz (Duke University). Spe I and Not I restriction sites were introduced using PCR. The full-length wild-type TRPV4 was subcloned into the PLVX-IRES-mCherry vector to generate TRPV4^WT (Clontech, Catalog No. 631237). To generate the TRPV4^FeRIC construct, PCR primers were designed to eliminate the 3′ stop site in wild-type TRPV4 and introduce a 3′ Not I site. PCR primers introducing a 5′ Not I site, and a 3′ Bam HI site and a stop codon were used to amplify human Kininogen1 domain 5 (FeRIC). This FeRIC fragment was subcloned into the Xba I and BamH 1 sites within the PLVX-IRES-mCherry vector containing TRPV4. All completed constructs were sequence-verified by the Molecular Cell Biology Sequencing Facility (UC Berkeley) and analyzed using MacVector 13.0 and Serial Cloner 2.6.

Hernández-Morales M., Han V., Kramer R.H, & Liu C. (2021). Evaluating methods and protocols of ferritin-based magnetogenetics. iScience, 24(10), 103094.

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CRISPR-Mediated Gene Knockout in Cell Lines

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This protocol was performed according to frequently used procedures^{16 (link)}. Target gene sgRNA oligonucleotides (GenScript, Nanjing, China) were cloned into the pLVX-IRES-mCherry vector (Clontech, CA, USA), and two different sgRNA guides were designed for each gene. Lipofectamine 3000 (Thermo Fisher) was used to transfect 1 µg of each plasmid into U87 MG cells, GL-261 cells or DCs. After 48 h, the transfection efficiencies of LGALS9^−/−, Rab27a^−/−, and nSMase2^−/− U87 MG cells and TIM3^−/− DCs were detected by measuring mCherry + fluorescence intensity, and the protein levels in the cells were detected by Western blotting or flow cytometry to determine the knockout efficiency. The sgRNA sequences are shown in Supplementary Table S3.

Wang M., Cai Y., Peng Y., Xu B., Hui W, & Jiang Y. (2020). Exosomal LGALS9 in the cerebrospinal fluid of glioblastoma patients suppressed dendritic cell antigen presentation and cytotoxic T-cell immunity. Cell Death & Disease, 11(10), 896.

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Cbln1 Promoter Luciferase Assay

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The Cbln1 promoter luciferase construct was purchased from GeneCopoeia, Inc. (Cat# MPRM22597-PG02), which encodes Gaussia luciferase driven by a 1331 bp genomic fragment upstream of the coding sequence of the murine Cbln1 gene (Accession: NC_000074.6). The Ube3a expression construct was generated by amplifying the coding sequence of human Ube3a isoform III from Plasmid #37605 (Addgene) with primers 5’-TCTTCCACTAGTGCCACCATGGCCACAGCTTGTAAAAGATC-3’ and 5’-TCTTCCGGATCCTTACAGCATGCCAAATCCTTTGG-3’ and subcloning into the SpeI and BamHI sites of the pLVX-IRES-mCherry vector (Clontech Cat#631237). HEK293T cells were transfected in 96-well using Lipofectamine 3000 Reagent (ThermoFisher) with 50 ng Cbln1 promoter-Gluc plasmid and 50 ng of either pLVX-IRES-mCherry or pLVX-Ube3a-IRES-mCherry per well (n = 6 each condition). 48 hours after transfection, Gaussia luciferase activity was developed using the Secrete-Pair Gaussia Luciferase Assay Kit (Genecopoeia, Inc., Cat#LF061) and measured with the BioTek Synergy 2 luminescence plate reader.

Nong Y., Stoppel D.C., Johnson M.A., Boillot M., Todorovic J., Shen J., Zhou X., Nadler M.J., Rodriguez C., Huo Y., Nagakura I., Kasper E.M, & Anderson M.P. (2023). UBE3A and transsynaptic complex NRXN1-CBLN1-GluD1 in a hypothalamic VMHvl-arcuate feedback circuit regulates aggression. bioRxiv.

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Inducible and Constitutive Gene Silencing Vectors

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Inducible shRNAs against TP53 and Trp53 (human and murine p53) or renilla (R) were cloned into the TRMPV-Neo vector (pSIN-TRE-dsRed-miR30-PGK-Venus-IRES-NeoR) as previously described (Weissmueller et al., 2014 (link), Zuber et al., 2011 (link)). Constitutive expression of shRNAs against polyC exons for the isoforms were cloned into the pSicoR vector (Addgene, 11579) as previously described (Ventura et al., 2004 (link)). Constitutive expression of shRNAs against hnRNPK were cloned into the GIPZ Lentiviral shRNA vector (Dharmacon). shRNA sequences are described in Key Resource Table.Constitutive expression of cDNAs encoding for +polyC and −polyC GAPs and indirect regulators of GTPase signaling shRNAs were cloned into the pLVX-IRES-mCherry vector (Clontech 631237). Constitutive expression of cDNAs encoding for human TP53 wild-type and mutants (R175H; R248Q and R273H) were cloned into the pLVX-IRES-PURO vector (Modified from Clontech 631237). All constructs were verified by sequencing. Lentiviruses and retroviruses were produced by transiently transfecting shRNAs or cDNA constructs using the Dharmacon Trans-Lentiviral shRNA Packaging Kit with Calcium Phosphate Transfection Reagent protocols into 293T cells and harvesting viral supernatants 48 h after transfection.

Escobar-Hoyos L.F., Penson A., Kannan R., Cho H., Pan C.H., Singh R.K., Apken L.H., Hobbs G.A., Luo R., Lecomte N., Babu S., Pan F.C., Alonso-Curbelo D., Morris JP I.V., Askan G., Grbovic-Huezo O., Ogrodowski P., Bermeo J., Saglimbeni J., Cruz C.D., Ho Y.J., Lawrence S.A., Melchor J.P., Goda G.A., Bai K., Pastore A., Hogg S.J., Raghavan S., Bailey P., Chang D.K., Biankin A., Shroyer K.R., Wolpin B.M., Aguirre A.J., Ventura A., Taylor B., Der C.J., Dominguez D., Kümmel D., Oeckinghaus A., Lowe S.W., Bradley R.K., Abdel-Wahab O, & Leach S.D. (2020). Altered RNA splicing by mutant p53 activates oncogenic RAS signaling in pancreatic cancer. Cancer cell, 38(2), 198-211.e8.

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