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Pmscv ires mcherry fp

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The PMSCV-IRES-mCherry FP is a plasmid that contains the IRES (Internal Ribosome Entry Site) sequence and the mCherry fluorescent protein gene. This plasmid can be used for bicistronic expression, allowing the translation of two separate proteins from a single mRNA transcript.

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Lab products found in correlation

Pcdna3 ova, by Addgene (2 mentions) Pmxs ires puro, by Cell Biolabs (1 mentions) Pmscv loxp dsred loxp egfp puro wpre, by Addgene (1 mentions) Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions) Nebuilder hifi dna assembly, by New England Biolabs (1 mentions)

6 protocols using pmscv ires mcherry fp

1

Retroviral Plasmid Construction and Transduction

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The following plasmids were utilized: pMSCV-IRES GFP9 (link), pMXs-IRES-Puro (Cell Biolabs Inc), pMSCV-IRES-mCherry FP (a gift from Dario Vignali, Addgene #52114), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE (a gift from Hans Clevers, Addgene #32702). The CSF3RT618I mutation was generated previously9 (link). Full-length CEBPA and JAK3 cDNA was obtained from Genecopia. The CEBPA and JAK3 mutations were generated using the Quikchange Site Directed Mutagenesis Kit (Agilent) using the primers in Supplementary Table 1. To produce retrovirus, 293T17 cells were transfected with EcoPac helper plasmid (a gift from Dr. Rick Van Etten) and the appropriate transfer plasmid. Conditioned media was harvested 48–72 h after transduction.
Braun T.P., Okhovat M., Coblentz C., Carratt S.A., Foley A., Schonrock Z., Curtiss B.M., Nevonen K., Davis B., Garcia B., LaTocha D., Weeder B.R., Grzadkowski M.R., Estabrook J.C., Manning H.G., Watanabe-Smith K., Jeng S., Smith J.L., Leonti A.R., Ries R.E., McWeeney S., Di Genua C., Drissen R., Nerlov C., Meshinchi S., Carbone L., Druker B.J, & Maxson J.E. (2019). Myeloid lineage enhancers drive oncogene synergy in CEBPA/CSF3R mutant acute myeloid leukemia. Nature Communications, 10, 5455.
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2

Mammalian Overexpression Plasmids

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pcDNA3.1 + C-HA Trp53 (OMu22847) and pcDNA3.1 + C-FLAG Ppia (OMu14516), pcDNA3.1 + C-FLAG Ppie (OMu11106) plasmids used in mammalian over-expression experiments were purchased from Genscript. pMSCV-Bcl2-IRES mCherry plasmid used in mammalian over-expression experiments in Type D cells was generated by cloning Bcl2 cDNA PCR amplified from tetO-Bcl2-IRES-tdtomato (Addgene Plasmid #117857) into pMSCV-IRES-mCherry FP (Addgene Plasmid #52114) using NEBuilder HiFi DNA Assembly (New England Biolabs #E2621S). Plasmids used in this study are available upon request.
Acosta J., Li Q., Freeburg N.F., Murali N., Indeglia A., Grothusen G.P., Cicchini M., Mai H., Gladstein A.C., Adler K.M., Doerig K.R., Li J., Ruiz-Torres M., Manning K.L., Stanger B.Z., Busino L., Murphy M., Wan L, & Feldser D.M. (2023). p53 restoration in small cell lung cancer identifies a latent cyclophilin-dependent necrosis mechanism. Nature Communications, 14, 4403.
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3

Construction of pMSCV-XBP1s-IRES-mCherry Vector

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To generate an pMSCV-XBP1s-IRES-mCherry construct, Flag-XBP1s was amplified from Flag-XBP1s-pcDNA5/FRT/TO (gift from Prof. David Ron) by PCR (F primer: cgccggaattcagatcttacgtagctagcgCAAATGGACTACAAAGACGA, R primer: gcggaattgatcccgctcgagcaattggTTAGACACTAATCAGCTGGG). This Flag-XBP1s fragment was integrated with pMSCV-IRES-mCherry fragment of pMSCV-IRES-mCherry FP (Addgene #52114, gift from prof. Dario Vignali) cut by bamHI.
Pramanik J., Chen X., Kar G., Henriksson J., Gomes T., Park J.E., Natarajan K., Meyer K.B., Miao Z., McKenzie A.N., Mahata B, & Teichmann S.A. (2018). Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation. Genome Medicine, 10, 76.
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4

Plasmid Construct Generation for Cellular Studies

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pBABE hygro MEN1 WT was a gift from Matthew Meyerson (Addgene 11024)85 (link). pCDH-EF1-Puro lentiviral vectors encoding HA-FLAG-tagged wildtype H3.3 and H3.3 K27M were a kind gift from Peter Lewis (University of Wisconsin)86 . All plasmids were verified by Sanger sequencing analysis through the Australian Genome Research Facility (Melbourne, Victoria). An ovalbumin (OVA) expressing retroviral vector was generated by PCR amplification of full-length chicken OVA from pcDNA3-OVA (Addgene 64599, a gift from Sandra Diebold & Martin Zenke)87 (link) and cloned into both pMSCV-IRES-mCherry FP (a gift from Paul Beavis) and pMSCV-IRES-GFP (modified from pMSCV-IRES-mCherry FP) via EcoRI and XhoI sites.
Sparbier C.E., Gillespie A., Gomez J., Kumari N., Motazedian A., Chan K.L., Bell C.C., Gilan O., Chan Y.C., Popp S., Gough D.J., Eckersley-Maslin M.A., Dawson S.J., Lehner P.J., Sutherland K.D., Ernst P., McGeehan G.M., Lam E.Y., Burr M.L, & Dawson M.A. (2023). Targeting Menin disrupts the KMT2A/B and polycomb balance to paradoxically activate bivalent genes. Nature cell biology, 25(2), 258-272.
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5

Plasmid Construct Generation for Cellular Studies

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pBABE hygro MEN1 WT was a gift from Matthew Meyerson (Addgene 11024)85 (link). pCDH-EF1-Puro lentiviral vectors encoding HA-FLAG-tagged wildtype H3.3 and H3.3 K27M were a kind gift from Peter Lewis (University of Wisconsin)86 . All plasmids were verified by Sanger sequencing analysis through the Australian Genome Research Facility (Melbourne, Victoria). An ovalbumin (OVA) expressing retroviral vector was generated by PCR amplification of full-length chicken OVA from pcDNA3-OVA (Addgene 64599, a gift from Sandra Diebold & Martin Zenke)87 (link) and cloned into both pMSCV-IRES-mCherry FP (a gift from Paul Beavis) and pMSCV-IRES-GFP (modified from pMSCV-IRES-mCherry FP) via EcoRI and XhoI sites.
Sparbier C.E., Gillespie A., Gomez J., Kumari N., Motazedian A., Chan K.L., Bell C.C., Gilan O., Chan Y.C., Popp S., Gough D.J., Eckersley-Maslin M.A., Dawson S.J., Lehner P.J., Sutherland K.D., Ernst P., McGeehan G.M., Lam E.Y., Burr M.L, & Dawson M.A. (2023). Targeting Menin disrupts the KMT2A/B and polycomb balance to paradoxically activate bivalent genes. Nature cell biology, 25(2), 258-272.
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6

Retroviral shRNA Expression Construct

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shRNAs were cloned into MSCV-PIG vector (from Dr. Mitchell Weiss) using Bgl II and Xho I sites. Sequences of oligonucleotides used to construct shRNA plasmids are described in Table S3B. shRNA-PuroR-IRES fragment was ligated into pMSCV-IRES-mCherry FP (Addgene, #52114) using Bgl II and Nco I sites. 293T cells transfected with 15 μg of pMSCV-PuroR-IRES-mCherry vector and pCL-Eco packaging vector produced retrovirus expressing shRNA. Cells were added to 100 μl viral supernatant, polybrene (8 mg/ml), and HEPES buffer, and spinoculated at 2,600 rpm for 90 min at 30°C.
Tanimura N., Liao R., Wilson G.M., Dent M.R., Cao M., Burstyn J.N., Hematti P., Liu X., Zhang Y., Zheng Y., Keles S., Xu J., Coon J.J, & Bresnick E.H. (2018). GATA/Heme Multi-omics Reveals a Trace Metal-Dependent Cellular Differentiation Mechanism. Developmental cell, 46(5), 581-594.e4.
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