The pLentiCRISPRv2-LoxP plasmid was generated by inserting LoxP sites (5′-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3′) flankingthe elongation factor-1 α short promoter of plentiCRISPRv2 (Addgene 52961; Shalem et al., 2014 (link)). gRNA sequences targeting lyspersin (5′-GCCGCAGCTTGACGTCTTCG-3′) and LAMTOR1 (5′-GAGGAGGGACGGCGTCCGTG-3′) were chosen using an online prediction tool (CRISPR Design; Zhang laboratory, MIT; Hsu et al., 2013 (link)) and subcloned into pLentiCRISPRv2-LoxP. HEK293LTV were cotransfected with the lentiviral plasmids pVSV-G (631530; Clontech) and psPAX2 (Vogel et al., 2015 (link)) and the viral supernatant directly used to infect HeLa cells. Transfected cells were subsequently selected with puromycin (P7255; Sigma-Aldrich). After selection, cells were transiently transfected with pCAG-Cre:GFP (13776; Addgene; Matsuda and Cepko, 2007 (link)) to remove the Cas9 and FACS sorted to enrich for the GFP-positive population. Limiting dilution then generated single cell clones. Finally, the KOs of lyspersin and LAMTOR1 (also referred to as LAMTOR1HM; de Araujo et al., 2017 (link)) were confirmed by PCR screen (Yu et al., 2014 (link)), Western blot analysis, and genotyping of the respective locus.
Pvsv g
Manufactured by Takara Bio
Sourced in United States
The PVSV-G is a plasmid that contains the VSV-G gene, which encodes the vesicular stomatitis virus glycoprotein. This plasmid is commonly used in lentiviral vector production.
Automatically generated - may contain errors
Lab products found in correlation
Pspax2, by Addgene (8 mentions)
Polybrene, by Merck Group (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Puromycin, by Merck Group (4 mentions)
Gp2 293 cells, by Takara Bio (4 mentions)
Puromycin, by Thermo Fisher Scientific (3 mentions)
Ultracel 50k filter centrifuge tubes, by Merck Group (3 mentions)
Pfastbac 1 transfer vector, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Antibiotic mixture, by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
X treme hp, by Roche (2 mentions)
Pretrox tight pur, by Takara Bio (2 mentions)
Pqcxip, by Takara Bio (2 mentions)
293ft cell, by Thermo Fisher Scientific (2 mentions)
Vsv g, by Merck Group (2 mentions)
Pqcxip retroviral vector, by Takara Bio (2 mentions)
Pqcxih, by Takara Bio (2 mentions)
Puromycin, by InvivoGen (2 mentions)
Pcag cre gfp, by Addgene (1 mentions)
43 protocols using pvsv g
1
Generation of CRISPR Knockout Cell Lines
2
Pseudotyped SIV Vectors for Inhibitor Screening
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SIV vectors bearing the VSV glycoprotein were prepared as indicated elsewhere [59 (link)]. Briefly, 293FT cells were transfected with 8 μg of plasmid coding for SIV Gag-Pol (pSIV3+), 8 μg of plasmid encoding GFP as an RNA minigenome (pGAE1.0) [60 (link)], and 4 μg of plasmid coding for the envelope protein G of VSV (pVSV-G, Clontech). Alternatively, the plasmid pI.18/GPC coding for ANDV glycoproteins was used to generate SIV vectors pseudotyped with ANDV glycoproteins. At 48 h post-transfection, supernatants containing pseudotyped particles were concentrated by centrifugation at 100,000 g for 75 min. Different dilutions of VSV-G pseudotyped SIV vectors were incubated for 1 h with Vero E6 cells in the presence and absence of protein or peptide inhibitor candidates. Three days later, the expression of GFP in transduced cells was analyzed by flow cytometry (FACScan, Becton Dickinson). ≥10.000 cells were counted for each experimental condition.
3
Construction of Retroviral Vectors for POLD1 Allele Expression
4
Lentivirus-Mediated Stable Gene Expression
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For lentivirus-mediated stable introduction of sfGFP-DARPin-LA6 and NLSSV40-sfCherry-NLSMyc, we followed the methods described previously (Liu et al., 2021 (link)). Briefly, pVSV-G (PT3343-5; Clontech) and psPAX2 (plasmid #12260; Addgene; http://n2t.net/addgene:12260 ; RRID:Addgene_12260; a gift from Didier Trono), together with the pCDH vector (pCDH-NLSSV40-sfCherry-NLSMyc-Blast or pCDH-sfGFP-DARPin-LA6-Hygro) in a 1:3:4 weight ratio of each plasmid was transfected into ∼80% confluent 293T cells (CRL-3216; ATCC) using Lipofectamine 3000 following the manufacturer’s instructions for lentivirus production. One day after the transfection, the medium was replaced with fresh medium, which was harvested at 48 h after transfection. For virus infection, MEFs, C2C12, BJ-5ta, and MCF10A cells were incubated with the virus-containing culture supernatants with 4 µg/ml polybrene (Nacalai Tesque) for 24 h. Infected cells were selected by incubation in medium containing 200 µg/ml hygromycin B Gold or 3 µg/ml blasticidin S (InvivoGen) for 4 d, except that C2C12 cells were selected with 20 µg/ml blasticidin S for 4 d.
5
Virus Propagation and Pseudotyping
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Ramos cells were propagated in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (GIBCO) and antibiotic mixture (Sigma) in a 5% CO2 atmosphere at 37°C. DT40 cells were propagated in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum and 1% chicken serum (GIBCO) and antibiotic mixture (Sigma) in a 5% CO2 atmosphere at 40°C. The AviPack packaging system was utilized for the ASLV-derived virus propagation and pseudotyping with vesicular stomatitis virus protein G (VSV-G) as described in Plachy et al. (2010) (link). HIV-derived vector was produced by 293T cell line co-transfection (X-Treme HP, Roche) with 1 μg of GFP7 vector, 1 μg of psPAX2 (Addgene plasmid # 12260) and 1 μg of pVSV-G (Clontech) in a 6 cm Petri dish. Viral supernatants were collected, filtered through a 0.45 μm SFCA filter and stored at −80°C.
6
Overexpression of CES2 in SU.86.86 Cancer Cells
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Parental SU.86.86 cells were purchased from ATCC. Manipulated SU.86.86 cell lines were provided by Dr Samir Hanash from MD Anderson Cancer Center and were cultured as previously described.11 (link) For overexpression, CES2 was cloned into pLenti-C-Myc-DDK-IRESPuro (OriGene, Rockville, MD) vector, an empty vector was used as a control. Lentiviral infections were performed using 293LTV cells (Cell Biolabs, Inc., San Diego, CA) as producers of viral supernatants. 293LTV cells were grown on 10 cm plates to 70% confluence and cotransfected with lentiviral DNA and the helper vectors pCMVΔ8.74 and pVSV-G (Clontech, Mountain View, CA) using Lipofectamine 3000 (Life Technologies), according to the manufacturer's instructions. The medium was harvested 48 h post-transfection, filtered through a 0.45 μm filter and used to cross-transduce cancer cells in the presence of 8 μg mL–1 Polybrene (Sigma-Aldrich). Subsequently clones were selected by puromycin. All cells were maintained in a 5% CO2 atmosphere at 37 °C. SU.86.86 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin/antimycotic (complete growth medium).
7
FOXO3A Overexpression in BaF3 Cells
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Production of retroviral particles and infection were performed as described above for lentiviral particles, but using 1.5 µg Hit60, 1.5 µg pVSV-G (Clontech) and either 1.5 µg pLXIN-hFOXO3A, plXIN-hFOXO3A-TM, or pLXIN-emtpy. Only BaF3 expressing lentiCRISPRv2 gRNA #1 targeting mouse Foxo3a were used, as human Foxo3a is also targeted by mouse Foxo3a gRNA #2. Selection was achieved with 50 ng/mL Geneticin™ (ThermoFisher, Waltham, MA, USA). Experiments were performed immediately after successful selection.
8
Retroviral Expression Plasmid Generation
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To generate retroviral expression plasmid pRetroX-Tight-Pur-SBP1, human SBP1 cDNA was amplified and subcloned into the pRetroX-Tight-Pur vector (Clontech, Mountain View, CA) using NotI and EcoRI restriction sites. The following primers were used: forward 5’- ATAGCGGCCGCTACAGCATGGCTACGAAAT-3’ and reverse 5’-ACGAATTCGCTCAAATCCA GATGTCAGAGC-3’ . Two plasmids pRetroX-Tet-On Advanced (Tet on activator vector) and pVSV-G (Packaging vector) were purchased from Clontech (Mountain View, CA).
9
Retroviral Expression Vectors for NSC-34 Cells
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To produce infecting retroviral expressing vectors, GP2-293 packaging cells (Clontech) were co-transfected with the above described TERC or alTERC constructs and with pVSV-G (Clontech). The medium containing the retroviral particles was used for the infection of NSC-34 cells. Cells containing the TERC or alTERC expressing vectors were selected by growing the infected NSC-34 cells with G418.
10
Lentiviral Transduction of SPARC shRNAs
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pLKO.1-Puro plasmids for control (shC002) and SPARC-targeting shRNAs TRCN0000008709 (sh8709) and TRCN0000008711 (sh8711) were from Sigma-Aldrich. Doxycycline-inducible SPARC-targeting V2THS_153399 (sh3399) was from Thermo. Each of these plasmids was co-transfected in HEK293T cells with pVSVG and pCMVΔR8.91 (Clontech, Mountain View, CA) using X-tremeGENE9 (Roche). Supernatants were collected for the following 48 h and filtered through 0.45 μm methylcellulose filters (Millipore). Viral particles were concentrated by ultracentrifugation on 20% sucrose gradients. Target cells were transduced in the presence of polybrene (8 μg/mL; Sigma-Aldrich), and selected with 1 μg/mL puromycin (Sigma-Aldrich) for 7 days.
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