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Plenti puro lentiviral vector

Manufactured by Addgene
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The PLenti-Puro lentiviral vector is a tool used for the delivery of genetic material into target cells. It provides a means to stably integrate a gene of interest into the host cell's genome, enabling long-term expression of the introduced genetic element. The vector utilizes a puromycin resistance cassette to allow for selection of transduced cells.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Plko 1 lentiviral vector, by Addgene (1 mentions) Pspax2, by Addgene (1 mentions) Pmd2 g, by Addgene (1 mentions) Plenti cmv tetr blast 716 1, by Addgene (1 mentions)

Spelling variants of this product

PLenti-puro (24 citations) PLenti-puro vector (13 citations) Lentiviral vector (10 citations) PLenti-CMV-LUC-Puro lentiviral vectors (2 citations) PLenti-CMV-puro lentiviral vector (2 citations)

3 protocols using plenti puro lentiviral vector

1

Lentiviral Knockdown and Overexpression

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shRNAs against Bmal1 (shBmal1#1 and #2), EBF3 (shEBF3) and ALOX15 (shALOX15) were provided by RiboBio and cloned into the pLKO.1 lentiviral vector (Addgene). Coding sequences for Bmal1, EZH2 and EBF3 were inserted into the pLenti‐puro lentiviral vector (Addgene). Lentiviral particles were packaged into HEK293T cells through transfection of pLenti‐puro‐Bmal1, pLenti‐puro‐EZH2, pLenti‐puro‐EBF3 or pLKO.1‐shBaml1, psPAX2 (Addgene) and pMD2.G (Addgene) using Lipofectamine 3000 (Thermo Fisher Scientific). After 72 h, lentiviral particles were collected and filtered. Subsequently, HL‐60 and NB4 cells were infected with lentiviral particles at a 100 multiplicity of infection (MoI). After 16 h, the medium was replaced with fresh medium, and cells with stable transfection were selected using puromycin.
Wang D., Wang F., Zhang H., Chen P, & Yang M. (2023). Circadian clock protein Bmal1 accelerates acute myeloid leukemia by inhibiting ferroptosis through the EBF3/ALOX15 axis. Cancer Science, 114(8), 3446-3460.
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2

Generating Lentiviral Constructs for NLRP3-CASP1 and ASC

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Plasmid of pLenti CMV TetR Blast (716-1) (#17492, Addgene, USA) was purchased from Addgene. pUC57 plasmid containing NLRP3-CASP1 gene (PUC57-CASP1-T2A-NLRP3-R260W), pUC57 plasmid contains ASC gene (pUC57-MYC-ASC) and CASPorter plasmid was synthesized and purchased from GenScript. CD514B-1/NLRP3-CASP1 construct was generated by inserting CASP1-T2A-NLRP3-R260W cDNA from PUC57-CASP1-T2A-NLRP3-R260W plasmid as a Nhel-BamHI fragment into the lentiviral backbone pCDH-CMV-MCS-EF1α-Neo/CD514B-1 (empty vector; System Biosciences, USA). pLenti-puro/myc-ASC construct was generated by inserting MYC-ASC cDNA from pUC57-MYC-ASC as an EcoRV-DraI fragment into the tetracycline-inducible pLenti-Puro lentiviral vector (#39481, Addgene, USA). The correctness of insertion was confirmed by digestion with EcoRV/DraI and DNA sequencing (Hartwell center, St. Jude Children’s Research Hospital, USA).
Zou C., Beard J.A., Yang G., Evans W.E, & Bonten E.J. (2022). CASPorter: A Novel Inducible Human CASP1/NALP3/ASC Inflammasome Biosensor. Journal of Inflammation Research, 15, 1183-1194.
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3

TurboID-Based Proximity Labeling

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TurboID and miniTurboID plasmids were obtained from Addgene (Plasmid #107171, #107172). Fragments of SWI/SNF subunits were subcloned from Addgene plasmids (SMARCD1: #21034; BICRAL/GLTSCR1L: #107732) and TransOMIC (BRD7: #BC094706; DPF2: #BC014889; PHF10: #BC020954). Fragments were cloned into TurboID plasmid to be in-frame with TurboID fusion and confirmed by sequencing. SMARCD1 with TurboID and mini-TurboID fusion was subcloned into pLenti-Puro lentiviral vector (Addgene: Plasmid #39481).
293T cells were transiently transfected with TurboID-fusion proteins, 48h post-transfection, cells were harvested for immunoprecipitation assay. G401 cells were infected with lentivirus (Turbo-SMARCD1 fusion or miniTurbo-SMARCD1), selected, and maintained with puromycin (1 μg/ml). We followed a TurboID benchmark study47 for biotin treatment, sample processing and data-analsysi. Briefly, biotin treatment time was optimized via streptavidin-IP followed by Western blotting for biotin. Optimal treatment times for TurboID and miniTurboID were 6 and 12 hours respectively (Fig. S2C). Cells were treated with Biotin (50 μM) and/or with Doxycycline with indicated times points and harvested for mass-spectrometry.
Wolf B.K., Zhao Y., McCray A., Hawk W.H., Deary L.T., Sugiarto N.W., LaCroix I.S., Gerber S.A., Cheng C, & Wang X. (2022). Cooperation of chromatin remodeling SWI/SNF complex and pioneer factor AP-1 shapes 3D enhancer landscapes. Nature structural & molecular biology, 30(1), 10-21.
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