Pmscvneo vector

Manufactured by Takara Bio

The PMSCVneo vector is a plasmid-based expression vector used for the production of recombinant proteins in mammalian cell lines. It contains the necessary elements for gene expression, including a promoter and a selection marker for antibiotic resistance.

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Lab products found in correlation

Pmscvneo, by Takara Bio (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Pvsv g plasmid, by Takara Bio (1 mentions) Pcdna4 hismax vector, by Thermo Fisher Scientific (1 mentions) Pcmv5 vector, by Takara Bio (1 mentions) Phusion high fidelity pcr master mix with hf buffer, by New England Biolabs (1 mentions) Gp2 293 packaging cells, by Takara Bio (1 mentions) Pacgfp1 n1 plasmid, by Takara Bio (1 mentions) Pfx polymerase, by Thermo Fisher Scientific (1 mentions) Pcr blunt 2 topo vector, by Thermo Fisher Scientific (1 mentions)

7 protocols using pmscvneo vector

Retroviral Vector Construction and Packaging

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Human INHBA DNA was prepared with RT-PCR using Pfx polymerase (Invitrogen), followed by subcloning into a pCR-Blunt II-TOPO vector (Invitrogen). The INHBA insert was then cut out, and subcloned into a pMSCVneo vector (Clontech). In order to prepare the expression construct for GFP-tagged BMPRII, DNA for whole-length human BMPRII was prepared by RT-PCR using Phusion High-Fidelity PCR Master Mix with HF Buffer (New England Biolabs), followed by subcloning into a pCR-Blunt II-TOPO vector (Invitrogen). The BMPRII insert was then cut out, and subcloned into a pAcGFP1-N1 plasmid (Clontech). Finally, the GFP-BMPRII insert was cut out and subcloned into the pMSCVneo vector (Clontech).
The INHBA/pMSCVneo and BMPRII-GFP/pMSCVneo constructs, alongside the pMSCVneo/GFP control construct, were transfected into GP2-293 packaging cells (Clontech) using a Lipofectamine 3000 (Thermo-Fisher) concurrent with a pVSVG viral envelope expression construct, followed by changing the medium with a fresh growth medium in 24 h. After an additional 24 h, the medium was changed again with a fresh growth medium and incubated for another 24 h. Subsequently, the culture medium containing retroviruses was collected and stored at −80 °C after the removal of floating cells by centrifugation.

Ryanto G.R., Ikeda K., Miyagawa K., Tu L., Guignabert C., Humbert M., Fujiyama T., Yanagisawa M., Hirata K.I, & Emoto N. (2021). An endothelial activin A-bone morphogenetic protein receptor type 2 link is overdriven in pulmonary hypertension. Nature Communications, 12, 1720.

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Retroviral transduction of myoblasts

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Preparation of retrovirus was performed as reported previously [11 (link)]. Briefly, cDNA for targeting gene (DARP-FLAG) was subcloned into pMSCVneo vector (Clontech). GP2-293 packaging cells were transfected with DARP-pMSCVneo plasmid and pVSV-G plasmid (Clontech) using Lipofectamine 3000. In a parallel way, GP2-293 cells were transfected with GFP-pMSCVneo and pVSV-G plasmids to prepare viruses for negative control. Fresh growth medium was replaced 24 h after transfection, and cells were incubated for another 24h, followed by collection of the virus-containing culture medium. For infection, myoblasts of 50–60% confluency were incubated in the mixture of growth medium and the virus-containing culture medium at 1:1 ratio in the presence of 8 μg/ml polybrene for 24 h. Thereafter, cells were given fresh medium and incubated for 24–48 h followed by protein extraction.

Shimoda Y., Matsuo K., Kitamura Y., Ono K., Ueyama T., Matoba S., Yamada H., Wu T., Chen J., Emoto N, & Ikeda K. (2015). Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle. PLoS ONE, 10(9), e0138624.

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Cloning and Expression of Human Mpl

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A plasmid encoding human Mpl (identical to reference sequence IMAGE:100016416 and accession no. BC153092) was procured from DNASU and used as the template for amplification of the DNA sequence encoding Mpl, which was then cloned into the pMSCV-neo vector (Clontech) using the EcoRI and XhoI sites for expression of untagged Mpl or into the pcDNA-COX2-54 vector via LIC for expression as an N-terminal His₆-FLAG fusion.

Venkatesan A., Geng J., Kandarpa M., Wijeyesakere S.J., Bhide A., Talpaz M., Pogozheva I.D, & Raghavan M. (2021). Mechanism of mutant calreticulin-mediated activation of the thrombopoietin receptor in cancers. The Journal of Cell Biology, 220(7), e202009179.

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Construction of Fusion Protein Vectors

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The pMSCV-neo MLL-ENL, PME (7 (link)) and Bcl2-ires-E2A-HLF (17 (link)) vectors are described elsewhere. Various MLL fusion constructs were generated by restriction enzyme digestion or polymerase chain reaction (PCR)-based mutagenesis. The cDNAs were cloned into the pMSCV neo vector (for virus production) (Clontech, Mountain View, CA) or the pCMV5 vector (for transient expression). The expression vectors for FLAG-tagged GAL4 fusion proteins were constructed by PCR using pM (Clontech) as template and cloned into the pCMV5 vector. Various LEDGF constructs were generated by PCR-based mutagenesis and cloned into the pcDNA4 HisMax vector (Invitrogen, Carlsbad, CA).

Okuda H., Kawaguchi M., Kanai A., Matsui H., Kawamura T., Inaba T., Kitabayashi I, & Yokoyama A. (2014). MLL fusion proteins link transcriptional coactivators to previously active CpG-rich promoters. Nucleic Acids Research, 42(7), 4241-4256.

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Cloning and Characterization of ΔU3hNPC1-WT

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cDNA encoding human ΔU3hNPC1-WT construct was kindly provided by Dan Ory (Washington University, St Louis, MO). The NPC1 gene was subcloned into bicistronic retroviral plasmid, pMIEG3 using EcoR1 and Not1 restriction enzyme sites. pMIEG3 vector was generated from pMSCVneo vector (Clontech) in which the murine phosphoglycerate kinase (PKG) promoter and the Neomycin resistance (Neo^r) genes were replaced by IRES (Internal Ribosome Entry Site) and EGFP (enhanced green fluorescent protein) genes. ΔU3hNPC1-WT construct has four substitutions compared with a standard reference NPC1 sequence (NM_000271.4, Uniprot ID:O15118), so this WT sequence is named as WT-V²¹. The substitutions are 387T>C (Y129Y), 1415T>C (L472P), 1925T>C (M642T), and 2587T>C (S863P). ΔU3hNPC1-WT has been used as a WT construct in precious publications^{21 (link),75 (link)}. It was derived from pSV-SPORT/NPC1 that has been used as the WT control protein in previous publications^{56 (link),76 (link),77 (link)}. The site-mutagenesis was generated by Quick-Change XL Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) using pMIEG3-NPC1 as template, and all the variants used in this study contain the four background variants derived from human ΔU3hNPC1-WT (i.e., WT-V) construct.

Wang C., Scott S.M., Subramanian K., Loguercio S., Zhao P., Hutt D.M., Farhat N.Y., Porter F.D, & Balch W.E. (2019). Quantitating the epigenetic transformation contributing to cholesterol homeostasis using Gaussian process. Nature Communications, 10, 5052.

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Multifaceted ASXL1 and BAP1 Expressions

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We used ASXL1-WT, ASXL1-MT, ASXL1-G646WfsX12, and ASXL1-MT-K351R in the pMYs-IRES-GFP (pMYs-IG) vector^{22 (link)} or pcDNA3.1 vector. We also used 3xFLAG-tagged, and Myc-tagged ASXL1-MT, FLAG-tagged and Myc-tagged ASXL1-MT-K351R, FLAG-tagged ASXL1-Y591X and Myc-tagged ASXL1-WT in the pMYs-IRES-GFP vector and HA-tagged ASXL1-MT in the pMYs-IRES-Blastcidin vector. For BAP1 expression, we used pEF-4xHA-BAP1 WT, pMMP-puro-BAP1 WT, and pMMP-puro-BAP1 C91S [gifts from Dr. Machida^{59 (link)}], and HA-tagged BAP1 WT/C91S in pMYs-IRES-nerve growth factor receptor (NGFR) vector and pMYs-IRES-tdTomato vector. For UBE2O expression, we used pLV-Myc-UBE2O WT and pLV-Myc-UBE2O C885S [gifts from Dr. Dijke^{60 (link)}]. For RUNX1-ETO expression, we used HA-tagged RUNX1-ETO or RUNX1-ETO9a in a pMSCV-IRES-Thy1.1 retroviral vector^{54 (link)}. For MLL-AF9 expression, we used pMSCV-MLL-AF9-pgk-EGFP retroviral vector^{54 (link)}. For HOXA7 and HOXA9 expression, we used Flag-tagged HOXA7 and HOXA9 in pMSCV-neo retroviral vector. The full-length cDNAs of HOXA7 and HOXA9 were purchased from Promega, and we cloned them into the pMSCV-neo vector (Clontech).

Asada S., Goyama S., Inoue D., Shikata S., Takeda R., Fukushima T., Yonezawa T., Fujino T., Hayashi Y., Kawabata K.C., Fukuyama T., Tanaka Y., Yokoyama A., Yamazaki S., Kozuka-Hata H., Oyama M., Kojima S., Kawazu M., Mano H, & Kitamura T. (2018). Mutant ASXL1 cooperates with BAP1 to promote myeloid leukaemogenesis. Nature Communications, 9, 2733.

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Retroviral Transduction of Human Cells

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Aff1 cDNA was ligated to 5’ KMT2A (Residue 1362) and cloned into the pMSCV neo vector (Clontech). Retroviruses for human cells were produced by transient transfection of 293T cells with viral plasmids along with envelope RD114 and the gag-pol M57 plasmids using the calcium-phosphate method. Retrovirus transduction to the cells was performed by spin infection at 3500 rpm for 4 h.

Isobe T., Takagi M., Sato-Otsubo A., Nishimura A., Nagae G., Yamagishi C., Tamura M., Tanaka Y., Asada S., Takeda R., Tsuchiya A., Wang X., Yoshida K., Nannya Y., Ueno H., Akazawa R., Kato I., Mikami T., Watanabe K., Sekiguchi M., Seki M., Kimura S., Hiwatari M., Kato M., Fukuda S., Tatsuno K., Tsutsumi S., Kanai A., Inaba T., Shiozawa Y., Shiraishi Y., Chiba K., Tanaka H., Kotecha R.S., Cruickshank M.N., Ishikawa F., Morio T., Eguchi M., Deguchi T., Kiyokawa N., Arakawa Y., Koh K., Aoki Y., Ishihara T., Tomizawa D., Miyamura T., Ishii E., Mizutani S., Wilson N.K., Göttgens B., Miyano S., Kitamura T., Goyama S., Yokoyama A., Aburatani H., Ogawa S, & Takita J. (2022). Multi-omics analysis defines highly refractory RAS burdened immature subgroup of infant acute lymphoblastic leukemia. Nature Communications, 13, 4501.

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