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Plvx ef1alpha ires mcherry vector

Manufactured by Takara Bio
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The PLVX-EF1alpha-IRES-mCherry Vector is a lentiviral expression vector that allows for the simultaneous expression of a gene of interest and the mCherry fluorescent reporter protein. The vector utilizes the EF1alpha promoter to drive gene expression and the IRES sequence to facilitate bicistronic mRNA translation.

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Lab products found in correlation

Celltiter 96 aqueous one solution cell proliferation assay, by Promega (2 mentions) Hct116, by American Type Culture Collection (2 mentions) Noble agar, by Merck Group (2 mentions) α tubulin, by Cell Signaling Technology (2 mentions) Thp 1, by American Type Culture Collection (2 mentions) Mtt reagent, by Promega (2 mentions) Hl 60, by American Type Culture Collection (2 mentions) Oci aml3, by Leibniz Institute DSMZ (2 mentions) Myd88, by Cell Signaling Technology (2 mentions) Anti il 8 antibody, by Abcam (2 mentions) Myd88 knockdown constructs, by Merck Group (2 mentions) Pd08959, by Selleck Chemicals (2 mentions) Cd11b and cd14 antibodies, by Thermo Fisher Scientific (2 mentions) Sp600125, by Selleck Chemicals (2 mentions) Sb203580, by Selleck Chemicals (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Pspax2 plasmid, by Addgene (1 mentions) Pmd2 g plasmid, by Addgene (1 mentions) Lenti x 293t cells, by Takara Bio (1 mentions)

Spelling variants of this product

PLVX vector (22 citations) PLVX-IRES-mCherry (16 citations) PLVX-IRES-mCherry vector (4 citations) PLVX-EF1alpha-IRES-mCherry (2 citations)

6 protocols using plvx ef1alpha ires mcherry vector

1

Multicolor Assay for Selective Advantage

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Selective advantage was performed through multicolor competition assay as previously described (Smogorzewska et al., 2007 (link); Zhang et al., 2016 (link)). Fibroblasts were transduced with a RAD50-IRES–mCherry expressing lentivirus vector (pLVX-EF1alpha-IRES-mCherry Vector, Takara Bio) or mCherry-expressing vector and treated or not with 100 ng/ml phleomycin for up to 18 days. The number of mCherry-positive cells was scored at various time points to assess for relative selective advantage of transduced (mCherry+) over untransduced (mCherry-) cells.
Chansel-Da Cruz M., Hohl M., Ceppi I., Kermasson L., Maggiorella L., Modesti M., de Villartay J.P., Ileri T., Cejka P., Petrini J.H, & Revy P. (2020). A Disease-Causing Single Amino Acid Deletion in the Coiled-Coil Domain of RAD50 Impairs MRE11 Complex Functions in Yeast and Humans. Cell Reports, 33(13), 108559.
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2

Complementation of Phleomycin Sensitivity

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For complementation of phleomycin sensitivity, patients’ SV40-transformed fibroblasts were transduced with a wtRAD50-IRES–mCherry expressing lentivirus vector (pLVX-EF1alpha-IRES-mCherry Vector, Takara Bio) or empty mCherry-expressing vector. Cells were cell sorted thanks to mCherry expression to obtain 100% of transduced cells. Sensitivity assays were then conducted as described above.
Chansel-Da Cruz M., Hohl M., Ceppi I., Kermasson L., Maggiorella L., Modesti M., de Villartay J.P., Ileri T., Cejka P., Petrini J.H, & Revy P. (2020). A Disease-Causing Single Amino Acid Deletion in the Coiled-Coil Domain of RAD50 Impairs MRE11 Complex Functions in Yeast and Humans. Cell Reports, 33(13), 108559.
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3

Lentiviral-mediated IKBKB Expression

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For reconstruction of WT or the mutant IKBKB expression to the IKBKB knocked out Jurkat E6–1 clone, the WT or mutant IKBKB cDNA was cloned into the pLVX-EF1alpha-IRES-mcherry Vector (TAKARA Bio USA, CA, USA). Lenti-X 293 T cells (TAKARA Bio USA) were plated into a T75 flask at a density of 9.4 × 106 cells overnight. The cells were transfected using Lipofectamine 3000 (Invitrogen) following the manufacture’s protocol with 10 μg of the pLVX-EF-1alpha IKBKB-IRES-mcherry vector or 10 μg of the pLVX-EF-1alpha IRES-mcherry vector, and two helper plasmids as 10 μg of psPAX2 plasmid (Addgene) and 1 μg of pMD2.G plasmids(Addgene). At 48 h after transfection, viruses were collected from the supernatants and concentrated by 100-fold using Leni-X™ Concentrator (TAKARA Bio USA). The IKBKB knocked out Jurkat E6–1 clones were infected with 10 μl of the concentrated viruses in RPMI containing 10% fetal bovine serum and 10 μg/ml of polybrene by spinning down the cells at 32 °C for 90 min.
Sacco K., Kuehn H.S., Kawai T., Alsaati N., Smith L., Davila B., Bundy V., Kuhns D.B., Dobbs K., Delmonte O., Notarangelo L.D., Rosenzweig S.D, & Keller M.D. (2022). A Heterozygous Gain-of-Function Variant in IKBKB Associated with Autoimmunity and Autoinflammation. Journal of clinical immunology, 43(2), 512-520.
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4

Investigating TLR8 Signaling in AML

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R848, MyD88 knockdown constructs, and noble agar were from Sigma (St Louis, MO, USA). SB203580, PD08959, and SP600125 were obtained from Selleck Chem (Boston MA, USA). MTT reagent was obtained in the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Madison WI, USA). p-p38, MyD88, α-tubulin, and actin antibodies were obtained from Cell Signaling Technologies (Beverly MA, USA). GAPDH was from Santa Cruz (Carlsbad, CA, USA). CD11b and CD14 antibodies were obtained from EBiosciences (San Diego CA, USA). Anti-Il-8 antibody was obtained from Abcam (Cambridge MA, USA). OCI-AML3 was obtained from DSMZ (Braunschweig, Germany) and 293T, HCT116, HL60, THP-1, and U937 cells were obtained from ATCC (Manassas, VA, USA). Primary AML human bone marrow cells were obtained from the CWRU Hematopoietic Stem Cell Core Facility (Cleveland, OH, USA). The cDNA for TLR8 was cloned into the pLVX-EF1alpha-IRES-mCherry vector obtained from Clontech (Mountain View, CA, USA)
Ignatz-Hoover J.J., Wang H., Moreton S.A., Chakrabarti A., Agarwal M.K., Sun K., Gupta K, & Wald D.N. (2014). The role of TLR8 signaling in Acute Myeloid Leukemia Differentiation. Leukemia, 29(4), 918-926.
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5

Investigating TLR8 Signaling in AML

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R848, MyD88 knockdown constructs, and noble agar were from Sigma (St Louis, MO, USA). SB203580, PD08959, and SP600125 were obtained from Selleck Chem (Boston MA, USA). MTT reagent was obtained in the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Madison WI, USA). p-p38, MyD88, α-tubulin, and actin antibodies were obtained from Cell Signaling Technologies (Beverly MA, USA). GAPDH was from Santa Cruz (Carlsbad, CA, USA). CD11b and CD14 antibodies were obtained from EBiosciences (San Diego CA, USA). Anti-Il-8 antibody was obtained from Abcam (Cambridge MA, USA). OCI-AML3 was obtained from DSMZ (Braunschweig, Germany) and 293T, HCT116, HL60, THP-1, and U937 cells were obtained from ATCC (Manassas, VA, USA). Primary AML human bone marrow cells were obtained from the CWRU Hematopoietic Stem Cell Core Facility (Cleveland, OH, USA). The cDNA for TLR8 was cloned into the pLVX-EF1alpha-IRES-mCherry vector obtained from Clontech (Mountain View, CA, USA)
Ignatz-Hoover J.J., Wang H., Moreton S.A., Chakrabarti A., Agarwal M.K., Sun K., Gupta K, & Wald D.N. (2014). The role of TLR8 signaling in Acute Myeloid Leukemia Differentiation. Leukemia, 29(4), 918-926.
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6

TNFSF9 cDNA Cloning and Mutagenesis

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A cDNA of TNFSF9 (NM_003811.4) was obtained by RT-PCR from RNA of HUT-78 cells with the forward primer 5′-CAC​CAT​GGA​ATA​CGC​CTC​TGA​CGC-3′ and the reverse primer 5′-TTA​TTC​CGA​CCT​CGG​TGA​AGG​G-3′ and inserted into the pCR 2.1-TOPOTA vector (Invitrogen) according to manufacturer’s instructions. The c.419T>G (p.V140G) and c.419T>C (p.V140A) mutants were obtained with a Q5 Site-Directed Mutagenesis Kit (NEB) with the forward primer 5′-AAG​GCT​GGA​GGC​TAC​TAT​GTC​TTC-3′ and reverse primer 5′-GGC​CAC​CAC​CAG​CTC​CTT-3′, and forward primer 5′-AAG​GCT​GGA​GCC​TAC​TAT​GTC​TTC​TTT​CAA​C-3′ and reverse primer 5′-GGC​CAC​CAC​CAG​CTC​CTT-3′, respectively. Coding sequences were confirmed by Sanger sequencing and subcloned into a bicistronic lentiviral vector encoding the mCherry protein as a reporter gene (pLVX-EF1alpha-IRES-mCherry Vector; ClonTech).
Fournier B., Hoshino A., Bruneau J., Bachelet C., Fusaro M., Klifa R., Lévy R., Lenoir C., Soudais C., Picard C., Blanche S., Castelle M., Moshous D., Molina T., Defachelles A.S., Neven B, & Latour S. (2022). Inherited TNFSF9 deficiency causes broad Epstein–Barr virus infection with EBV+ smooth muscle tumors. The Journal of Experimental Medicine, 219(7), e20211682.
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