Pmcherry vector

Manufactured by Takara Bio

Sourced in United States

The PmCherry vector is a plasmid that expresses the mCherry fluorescent protein under the control of the CMV promoter. mCherry is a red fluorescent protein derived from DsRed that can be used as a reporter for gene expression or protein localization studies.

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Lab products found in correlation

Cuy21sc electroporator, by Nepa Gene (1 mentions) Hepes, by Merck Group (1 mentions) Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions) I scei, by New England Biolabs (1 mentions) Pacgfp vector, by Takara Bio (1 mentions) Gene pulser, by Bio-Rad (1 mentions)

Close spelling variants of this product

PmCherry-C1 mammalian expression vector PmCherry-C1 expression vector PmCherry-N1 vector PmCherry-C1 vector PmCherry-C2 vector PmCherry

5 protocols using pmcherry vector

Dual Fluorescent Transgenic Mice for Odf2 and Odf4

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All Tg mice were produced according to a previously reported method^{27 (link)}. Double-Tg(Odf2-mCherry,Odf4-Egfp) mice. First, we generated Tg(Odf4-Egfp)Odf4^+/+ mice and Tg(Odf2-mCherry)Odf4^+/+ mice separately (Fig. 1C). The mouse Odf4 cDNA was fused with a linearized pCM-Egfp vector containing the mouse Calmegin promoter^{28 (link)}, which was made from the pCM-Eqtn-Egfp vector^{27 (link)}. The mouse Odf2 cDNA was cloned into the pmCherry vector (Clontech), and then the Odf2-mCherry fragment was cloned into the pCM vector (pCM-Odf2-mCherry). The transgenes excised from pCM-Odf4-Egfp and pCM-Odf2-mCherry were injected into fertilized eggs of C57BL/6 mice. Incorporation of the transgene was detected via PCR analysis using primer set A for Tg(Odf4-Egfp) and primer set B for Tg(Odf2-mCherry), as shown in Table S2. Tg(Odf4-Egfp) mice were crossed with Tg(Odf2-mCherry) mice to generate double Tg(Odf2-mCherry,Odf4-Egfp) mice (Fig. 1C). Tg(Eqtn-Egfp)Odf4^+/+ mice were generated as previously reported^{27 (link)}. Tg(Eqtn-Egfp)Odf4^−/− was generated by crossing Tg(Eqtn-Egfp)Odf4^+/+ males with Odf4^−/− females. Rescue Tg males. Tg(Odf4-Egfp)Odf4^+/+ males were mated with Odf4^−/− females to obtain heterozygous Tg(Odf4-Egfp)Odf4^+/- mice. Tg(Odf4-Egfp)Odf4^+/- males and females were mated to obtain Tg(Odf4-Egfp)Odf4^−/− mice for the rescue experiment.

Ito C., Makino T., Mutoh T., Kikkawa M, & Toshimori K. (2023). The association of ODF4 with AK1 and AK2 in mice is essential for fertility through its contribution to flagellar shape. Scientific Reports, 13, 2969.

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Electroporation of Embryos with Fluorescent Proteins

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A pmCherry vector (Clontech, Mountain View, CA, USA) was transferred into a pCAGGS (N-R) plasmid [25 (link)] to produce protein under the control of a strong ubiquitous promoter based on the β-actin promoter. Fertilized embryos in the anterior nucleus stage were subjected to electroporation in 30 microL of HBS buffer [20 mM HEPES, pH 7.0–7.6 (Sigma-Aldrich, Saint Louis, MO, USA) and 150 mM NaCl] containing 45 microg of mCherry, siUNC5C with mCherry (ratio of 10:1), Rhox5 with mCherry, siG9a with mCherry, or siNetrin-1 with mCherry. siUNC5C and siNetrin-1 were generated and selected as shown in Table H in S1 File. Three sets of four electric pulses (21 V, duration of 1 ms, interval of 99 ms) were delivered using a CUY21SC electroporator (Nepagene, Chiba, Japan) [27 (link)].

Sugimoto M., Gotoh Y., Kawahara T, & Sugimoto Y. (2015). Molecular Effects of Polymorphism in the 3’UTR of Unc-5 homolog C Associated with Conception Rate in Holsteins. PLoS ONE, 10(7), e0131283.

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Plasmid-based Transfection Assay for DNA Repair

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Plasmids carrying NHEJ or HR reporter cassettes containing a GFP gene, as described [53 ], were linearized by I-SceI (NEB) and used for transfecting cells: 0.5 μg and 2 μg respectively. 0.1 μg of pmCherry vector (Clontech) was co-transfected in both cases as a transfection efficiency control. Transfections were performed using TurboFect transfection reagent (Fermentas). 48 hrs later cells were harvested and analysed by 2-laser FACS.

Lezina L., Aksenova V., Fedorova O., Malikova D., Shuvalov O., Antonov A.V., Tentler D., Garabadgiu A.V., Melino G, & Barlev N.A. (2015). KMT Set7/9 affects genotoxic stress response via the Mdm2 axis. Oncotarget, 6(28), 25843-25855.

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Molecular Cloning and Protein Expression

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ANFP-EmGFP was a generous gift from Dr Ed Levitan, University of Pittsburgh, and the insert subcloned into pmCherry vector (Clontech). Scinderin was directly cloned from mouse brain cDNA by RT-PCR, and inserted in-frame into the pAcGFP vector from Clontech. Mutations to convert it to a shRNA suppression resistant form, as well as PIP binding domain mutations, were introduced by standard PCR. All constructs were fully sequenced by CCHMC sequence center to ensure accuracy and reading frame. Protein expression and purification used the BioRad Profinity system. Protein concentrations were measured by BCA assay. Gene knockdown was targeted for the sequence identified in Figure 1, and inserted into the bi-cistronic LMP vector (a kind gift from Dr. Roger Briesewitz, Ohio State University), together with either GFP, or a GFP-shRNA resistant scinderin fusion. The cytoplasmic domain of synaptobrevin/VAMP (amino acids 1–91, cdVamp) was created by PCR from VAMP2 purchased from Open Biosystems, and cloned into the AcGFP vector to allow visualization of transfected cells. Transfections were carried out using electroporation in a Gene Pulser (Bio Rad) using 20 μg DNA.

Wang J, & Richards D.A. (2017). The actin binding protein Scinderin acts in PC12 cells to tether dense-core vesicles prior to secretion. Molecular and cellular neurosciences, 85, 12-18.

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Fluorescent Tagging of KCC2 Variants

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KCC2-IRES-GFP (Chudotvorova et al., 2005 (link)), eGFP-KCC2 (Pellegrino et al., 2011 (link)), and KCC2-pH_ext (Kahle et al., 2014 (link)) were provided by I. Medina. ΔNTD-KCC2 (Li et al., 2007 (link)) was obtained from C. Rivera. Nontagged wild-type KCC2 (WT-KCC2) was obtained by removal of the sequence encoding the eGFP tag from eGFP-KCC2. ΔNTD-KCC2-pH_ext mutant (deletion of 1–100 amino acids) was created using PCR. ΔCTD-KCC2 mutant (deletion of KCC2’s 654–1114 amino acids) was made by deleting the corresponding piece of KCC2 using BamHI endonuclease. mCherry construct was created by insertion of an ubiquitin promoter instead of CMV promoter in pmCherry vector (Clontech). All constructs were verified by DNA sequencing. More details on vectors and constructs are available on request.

Friedel P., Ludwig A., Pellegrino C., Agez M., Jawhari A., Rivera C, & Medina I. (2017). A Novel View on the Role of Intracellular Tails in Surface Delivery of the Potassium-Chloride Cotransporter KCC2. eNeuro, 4(4), ENEURO.0055-17.2017.

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