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Plv ef1a ires neo

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The PLV-EF1a-IRES-Neo is a plasmid vector that contains the EF1a promoter, an IRES sequence, and a neomycin resistance gene. It is designed for the expression of genes of interest in mammalian cell lines and can be used for selection of transfected cells with neomycin.

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Lab products found in correlation

E z n a endo free plasmid dna kit, by Omega Bio-Tek (2 mentions) Plv ef1a ires hygro, by Addgene (1 mentions) Str analysis, by Eurofins (1 mentions) Vero e6 cell, by American Type Culture Collection (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions) Gibson assembly kit, by New England Biolabs (1 mentions) Lenticrispr v2, by Addgene (1 mentions)

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PLV-EF1a-IRES-NEO vector (3 citations)

7 protocols using plv ef1a ires neo

1

Lentiviral Transduction of ACE2 and TMPRSS2

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The pLV-EF1a-IRES-Hygro (Addgene plasmid #85134) and pLV-EF1a-IRES-Neo (Addgene, plasmid #85139) lentiviral vectors were modified to encode synthesised (Integrated DNA Technologies) ACE2 (GenBank NM_001371415.1) and TMPRSS2 (GenBank NM_005656). Lentiviral vectors were produced through transient transfection as described previously [42 (link)] and used to modify A549 cells (ATCC#CCL-185; generous gift from Prof. Ben Hale, validated by STR analysis (Eurofins)) and Vero E6 cells (ATCC#CRL-1586; generous gift of Prof. Michele Bouloy) that were cultured and transduced under standard culture conditions.
Rihn S.J., Merits A., Bakshi S., Turnbull M.L., Wickenhagen A., Alexander A.J., Baillie C., Brennan B., Brown F., Brunker K., Bryden S.R., Burness K.A., Carmichael S., Cole S.J., Cowton V.M., Davies P., Davis C., De Lorenzo G., Donald C.L., Dorward M., Dunlop J.I., Elliott M., Fares M., da Silva Filipe A., Freitas J.R., Furnon W., Gestuveo R.J., Geyer A., Giesel D., Goldfarb D.M., Goodman N., Gunson R., Hastie C.J., Herder V., Hughes J., Johnson C., Johnson N., Kohl A., Kerr K., Leech H., Lello L.S., Li K., Lieber G., Liu X., Lingala R., Loney C., Mair D., McElwee M.J., McFarlane S., Nichols J., Nomikou K., Orr A., Orton R.J., Palmarini M., Parr Y.A., Pinto R.M., Raggett S., Reid E., Robertson D.L., Royle J., Cameron-Ruiz N., Shepherd J.G., Smollett K., Stewart D.G., Stewart M., Sugrue E., Szemiel A.M., Taggart A., Thomson E.C., Tong L., Torrie L.S., Toth R., Varjak M., Wang S., Wilkinson S.G., Wyatt P.G., Zusinaite E., Alessi D.R., Patel A.H., Zaid A., Wilson S.J, & Mahalingam S. (2021). A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research. PLoS Biology, 19(2), e3001091.
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2

Engineered Lentiviral Vectors for Gene Editing

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The SCRPSY (KT368137.1) lentiviral vector has been previously described [2 (link)], pLV-EF1a-IRES-Neo (Addgene plasmid #85139) was modified to include SfiI sites flanking the transgene ORF by inserting the TagRFP (or gene of interest) ORF with flanking SfiI sites between the unique BamHI and EcoRI restriction sites using PCR (primer pair AW177-BamHI-SfiI-RFP-F’ 5’-CTCTCGGATCCGGCCGAGAGGGCCATGAGCGAGCTGATTAAG-3’ and AW178-EcoRI-SfiI-RFP-R’ 5’-CTCTCGAATTCGGCCAGAGAGGCCTCACTTGTGCCCCAG-3’). Gene editing was achieved using the lentiCRISPRv2-Blast system [78 (link)].
Sugrue E., Wickenhagen A., Mollentze N., Aziz M.A., Sreenu V.B., Truxa S., Tong L., da Silva Filipe A., Robertson D.L., Hughes J., Rihn S.J, & Wilson S.J. (2022). The apparent interferon resistance of transmitted HIV-1 is possibly a consequence of enhanced replicative fitness. PLOS Pathogens, 18(11), e1010973.
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3

Engineered Light-Responsive TGF-β Signaling

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Retroviral plasmids containing tdTomato-tagged blue light-responsive TGF-β receptors (Addgene #118942) and iRFP682-tagged Smad2 (Addgene # 118943) were provided as a gift by Dr. Won Do Heo.24 (link) Transformed colonies of competent E. coli (C3019H; New England BioLabs) were selected, amplified, and purified (E.Z.N.A. Endo-Free Plasmid DNA Kit; Omega Bio-Tek). Omitting the retroviral backbone, lentiviral plasmids were designed using Benchling and SnapGene software and constructed using custom cloning service of Epoch Life Science. The sequences for the light-responsive TGF-β receptors and iRFP682-Smad2 were inserted separately into lentiviral backbones (pLV-EF1a-IRES-Neo; Addgene #85139). The lentiviral plasmids were similarly amplified and purified.
Wu J.Y., Yeager K., Tavakol D.N., Morsink M., Wang B., Soni R.K., Hung C.T, & Vunjak-Novakovic G. (2023). Directed differentiation of human iPSCs into mesenchymal lineages by optogenetic control of TGF-β signaling. Cell reports, 42(5), 112509.
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4

Lentiviral STING Overexpression

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A STING cDNA in the pUNO3 vector was obtained from InvivoGen (San Diego, CA). The STING ORF was PCR amplified with a 5′ BamHI and 3′ NotI restriction site and cloned into pLV-EF1a-IRES-Neo (Addgene # 85139). Lentivirus was produced by co-transfecting HEK293T cells with the STING ORF containing vector the packaging plasmids psPAX2 and pMD2.G (Addgene #12260 and 12259). STING-KO cells were transduced and selected with 800 µg/mL G418.
Hayman T.J., Baro M., MacNeil T., Phoomak C., Aung T.N., Cui W., Leach K., Iyer R., Challa S., Sandoval-Schaefer T., Burtness B.A., Rimm D.L, & Contessa J.N. (2021). STING enhances cell death through regulation of reactive oxygen species and DNA damage. Nature Communications, 12, 2327.
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5

Generation of PTEN Mutant iBMMs

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cDNA was prepared from wildtype iBMMs by extracting total RNA using TRIzol (Invitrogen) and reverse transcription with an iScript cDNA Synthesis Kit (Bio-Rad). PTEN coding sequence was PCR amplified from cDNA and cloned into pLV-EF1a-IRES-Neo (Addgene #85139) [81 (link)] using restriction cloning. To prevent CRISPR/Cas9-mediated genome editing in Pten–/– iBMMs which constitutively express Cas9 and Pten-specific sgRNA, synonymous point mutations were made in each amino acid within the sgRNA recognition site using site-directed mutagenesis to generate a CRISPR-resistant Pten allele (PTEN-CR). PTEN-CR was further mutagenized using site-directed mutagenesis to create G129E or G129R point mutations. Empty pLV-EF1a-IRES-Neo, pLV.PTEN-CR, pLV.PTEN-CR-G129E, or pLV.PTEN-CR-G129R were packaged into lentivirus and transduced into Pten–/– iBMMs or wildtype iBMMs (empty vector only) as described above. After antibiotic selection, protein expression of each PTEN variant was confirmed by immunoblot as described above.
Glover R.C., Schwardt N.H., Leano S.K., Sanchez M.E., Thomason M.K., Olive A.J, & Reniere M.L. (2023). A genome-wide screen in macrophages identifies PTEN as required for myeloid restriction of Listeria monocytogenes infection. PLOS Pathogens, 19(5), e1011058.
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6

Cloning and Lentiviral Transduction of INPPL1 and MAP4K5

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The open reading frames of INPPL1 (GenScript: OHu19348), MAP4K5 (GenScript: OHu02673), and various mutants of these genes were cloned into pLV-EF1a-IRES-Neo (85139; Addgene) using a Gibson Assembly kit (E5510S; NEB). All sgRNAs were cloned into lentiCRISPRv2 (52961; Addgene) by following a published protocol (Sanjana et al., 2014 (link); Shalem et al., 2014 (link)). Lentiviruses were produced by co-transfecting HEK293T cells with viral construct and packaging vector DNAs (Hart et al., 2017 (link); Mair et al., 2019 (link)). Cells were spin-infected with concentrated viruses and infected cells were recovered by selection with the appropriate antibiotics.
Wei W., Geer M.J., Guo X., Dolgalev I., Sanjana N.E, & Neel B.G. (2023). Genome-wide CRISPR/Cas9 screens reveal shared and cell-specific mechanisms of resistance to SHP2 inhibition. The Journal of Experimental Medicine, 220(5), e20221563.
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7

Engineered Light-Responsive TGF-β Signaling

Cited 1 time
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Retroviral plasmids containing tdTomato-tagged blue light-responsive TGF-β receptors (Addgene #118942) and iRFP682-tagged Smad2 (Addgene # 118943) were provided as a gift by Dr. Won Do Heo.24 (link) Transformed colonies of competent E. coli (C3019H; New England BioLabs) were selected, amplified, and purified (E.Z.N.A. Endo-Free Plasmid DNA Kit; Omega Bio-Tek). Omitting the retroviral backbone, lentiviral plasmids were designed using Benchling and SnapGene software and constructed using custom cloning service of Epoch Life Science. The sequences for the light-responsive TGF-β receptors and iRFP682-Smad2 were inserted separately into lentiviral backbones (pLV-EF1a-IRES-Neo; Addgene #85139). The lentiviral plasmids were similarly amplified and purified.
Wu J.Y., Yeager K., Tavakol D.N., Morsink M., Wang B., Soni R.K., Hung C.T, & Vunjak-Novakovic G. (2023). Directed differentiation of human iPSCs into mesenchymal lineages by optogenetic control of TGF-β signaling. Cell reports, 42(5), 112509.
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