Myristoylated-Akt1 (myrAkt1) lentivirus was generated by co-transfecting pCCL-myrAkt1 backbone15 (link) (13 μg) with RRE (5 μg), REV (2.5 μg), and VSV-G (3 μg) packaging plasmids on a 10 cm dish of 80% confluent 293T/17 cells (ATCC CRL-11268) using Lipofectamine 2000 (ThermoFisher Scientific 12566-014) according to the manufacturer’s suggestions. Forty-eight hours post-transfection, supernatants were processed using a Lenti-X Concentrator (ClonTech 631232) according to the manufacturer’s suggestions. Precipitated myrAkt1 lentivirus was resuspended in 0.5 mL TNE Buffer (50 mM Tris pH 8.0, 1 mM EDTA, 130 mM NaCl), aliquoted, and stored at −80 °C. Viral titers were determined using Lenti-X p24 Rapid Titer Kit (ClonTech 632200). IkB-SS expressing lentiviral vector was generated by sub-cloning the sequence of human IκBα super-suppressor (Addgene #15264) into pLVX Puro Vector (Clontech #632164). IkB-SS lentivirus was generated by co-transfecting pLVX-Puro-IkB-SS vector with RRE, REV, and VSV-G packaging plasmids in 293T cells as described earlier. pLVX Puro Vector (Clontech #632164) was utilized to generate the “Puro-empty” lentivirus.
Plvx puro vector
Manufactured by Takara Bio
Sourced in United States, Japan
The PLVX-Puro vector is a lentiviral expression vector that allows for the stable expression of genes of interest in target cells. The vector features a puromycin resistance gene, enabling the selection of transduced cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (18 mentions)
Polybrene, by Merck Group (8 mentions)
Pspax2, by Addgene (5 mentions)
Pmd2 g, by Addgene (5 mentions)
Plko 1 vector, by Addgene (5 mentions)
Plko 1, by Addgene (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Plko 1 puro vector, by Addgene (3 mentions)
Site directed mutagenesis kit, by Thermo Fisher Scientific (3 mentions)
Pcdna3, by Takara Bio (3 mentions)
Plvx puro vector, by Addgene (2 mentions)
Lenti x p24 rapid titer kit, by Takara Bio (2 mentions)
Puromycin, by Merck Group (2 mentions)
Pegfp c1, by Takara Bio (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Human gatad2a orf, by GenScript (2 mentions)
Phr sffv dcas9 bfp krab, by Addgene (2 mentions)
Lenti x packaging single shots vsv g system, by Takara Bio (2 mentions)
Fast trap lentivirus purification and concentration kit, by Merck Group (2 mentions)
293t cell, by American Type Culture Collection (2 mentions)
98 protocols using plvx puro vector
1
Generation of Myristoylated-Akt1 and IκB-SS Lentiviruses
2
Cloning and Transfection of USP49 and PPMIA
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The USP49 (AJ586139.1) gene was cloned into pLVX-Puro vector (Clontech) using EcoRI and BamHI with primers (Table 1).
The PPMIA gene was cloned into pLVX-Puro vector (Clontech) using EcoRI and BamHI with primers (Table 1).
Cell culture and transfection 293T cells were purchased from American Type Culture Collection (ATCC, VA, USA)
and were cultured with DMEM (Gibco, CA, USA) supplemented with 10% FBS. Cells were cultured at 37℃ under a humidified 5% CO2.
The pLVX-Puro-USP49, psPAX2, and pMD2G (Addgene) were cotransfected into 293T cells using Lipofectamine™ 2000 (Invitrogen, CA, USA). Then, cells were cultured in a complete medium after 6-hour incubation, and lentiviruses were harvested at 48 hours and 72 hours. HK-2 cells were transfected with 1.5 μg of pLVX-Puro-USP49 using Lipofectamine 2000 reagent. Cells transfected with pLVX-Puro were used as the control.
The PPMIA gene was cloned into pLVX-Puro vector (Clontech) using EcoRI and BamHI with primers (Table 1).
Cell culture and transfection 293T cells were purchased from American Type Culture Collection (ATCC, VA, USA)
and were cultured with DMEM (Gibco, CA, USA) supplemented with 10% FBS. Cells were cultured at 37℃ under a humidified 5% CO2.
The pLVX-Puro-USP49, psPAX2, and pMD2G (Addgene) were cotransfected into 293T cells using Lipofectamine™ 2000 (Invitrogen, CA, USA). Then, cells were cultured in a complete medium after 6-hour incubation, and lentiviruses were harvested at 48 hours and 72 hours. HK-2 cells were transfected with 1.5 μg of pLVX-Puro-USP49 using Lipofectamine 2000 reagent. Cells transfected with pLVX-Puro were used as the control.
3
Stable Overexpression of TDO2 in ESCC Cell Lines
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The stable overexpression of TDO2 in KYSE150 and KYSE450 and detailed experiments were performed as described previously28 (link). Briefly, full-length TDO2 cDNA (Supporting Information Table S3 ) extracted from ESCC tumor was amplified by PCR using PrimeSTAR HS DNA Polymerase (TaKaRa, Japan), and subcloned into XhoI and BamHI sites of pLVX-puro vector (Clonetech) to produce lentivirus in HEK-293T. KYSE150 and KYSE450 cells were transfected with concentrated virus using Lipofectamine 2000 (Invitrogen) and then cultured for 48 h, followed by puromycin selection (0.1 mg/mL, Sigma). Cells transfected with empty vector were used as negative control.
4
Fluorescently-tagged and biotinylatable human γTuRC production
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To generate a fluorescently-tagged and biotinylatable human γTuRC, the coding region for full-length human GCP2 (amino acids 1-902) was amplified by PCR using its cDNA as template (NM_001256617.1, Origene). The mTagBFP (blue fluorescent protein, Evrogen) coding sequence was also amplified by PCR. Both PCR-amplified sequences were cloned into a pLVX-Puro vector (Clonetech) using Gibson assembly (In-Fusion cloning, Takara), to form GCP2_G5A_TEV_G5A_mTagBFP_G5A_BAP, an expression construct for GCP2 which is C-terminally tagged with mTagBFP and biotin acceptor peptide (BAP: GLNDIFEAQKIEWHE), both separated from GCP2 by a TEV protease cleavage site. Glycine linkers (G5A) were placed between sequences. To facilitate the in vivo biotinylation of tagged γTuRC E. coli biotin ligase BirA was cloned into a pLVX-IRES-Hyg vector (Clonetech) using Gibson assembly to form HA_ G5A_BirA; an expression construct of BirA with an HA-tag added to the BirA N-terminus, separated by a G5A-linker. Primers used for cloning are listed in the Key Resources Table .
5
Manipulating NF-kB Signaling in ccRCC
6
Lentiviral Transduction of GFP-tagged VEGFR2
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Human VEGFR2 cDNA was modified to replace the signal peptide with a strong IgG kappa signal peptide followed by enhanced GFP. The resultant mature protein has GFP fused at the natural VEGFR2 N-terminus. This construct was subcloned into the pLVXpuro vector (Takara Bio). Lentiviral particles were produced in a HEK293T packaging line, using standard methods. Briefly, cells were transfected with pLVXpuro and the packaging vectors pMDG2 and psAX2, using polyethylenimine. After 48 h, the cell medium was added to CiGEnC cells overnight. The following day, the process was repeated with fresh virus. Cells were selected for stable expression in media supplemented with 2 µg/ml puromycin.
7
Lentiviral Transduction of Mesenchymal Stem Cells
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Construct pLVX‐Puro‐CiC was generated as described before (Pouikli et al, 2021 ) by cloning the CiC cDNA sequence fused to C‐terminal FLAG epitope (synthesized by Geneart) into a pLVX‐Puro vector (Takarabio) between the XhoI and XbaI restriction sites. Lentivirus was generated by co‐transfection of HEK293 T cells, cultured in control DMEM medium (CDM; DMEM‐GlutaMAX, 10% FBS and 1× penicillin/streptomycin) with the pLVX‐Puro vector or the pLVX‐Puro‐Slc25a1‐FLAG, pMD2.G and psPAX2 vectors (the pMD2.G and psPAX2 were a gift from D. Trono; Addgene plasmid catalogue nos. 12259 and 12260, respectively). Specifically, 2.8 million HEK cells were seeded in a 10 cm plate. The next day, a transfection mix of 500 ml 2 × HBS, 62 ml CaCl2 2 M, 10 mg pLVX, 5.2 mg pMD2.G and 5.2 mg psPAX2 was incubated for 30 min at RT and subsequently added to the cells. After 16 h, the medium was changed to 6 ml fresh CDM. After 72 h, the supernatant was collected, spun at 500 g for 5 min and filtered through a 0.45 μm filter.
A 1:1 virus:medium ratio along with 4 μg/ml polybrene was used to transduce MSCs. The medium was changed after 18 h. Puromycin (2 μg/ml) was added after 72 h to positively select transduced cells.
A 1:1 virus:medium ratio along with 4 μg/ml polybrene was used to transduce MSCs. The medium was changed after 18 h. Puromycin (2 μg/ml) was added after 72 h to positively select transduced cells.
8
Genetic Engineering of Immune Proteins
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PDCD1 was cloned from human T cell cDNA. The dominant-negative Src homology region 2 domain-containing phosphatase-2 (SHP2) mutant (SHP2CS) was a gift from Chengcheng Zhang Lab (UT Southwestern Medical Center). SHP2CS was cloned into the pLVX-IRES-ZsGreen1 vector (Takara Bio Cat# 632187). PDCD1 was fused with human Fc and cloned into the plasmid pFuse-hIgG1-Fc1 (InvivoGen). Mouse galectin-7 (LGALS7) cDNA (VectorBuilder) was cloned into a pLVX-puro vector (Takara Bio #632164). The PD-1 glycosylation site mutants (N49Q, N58Q, N74Q, N116Q) were made with Q5 site-directed mutagenesis kit (New England Biolabs Cat#E0554S), using the wild type constructs (pFuse-PD1-Fc or pLVX-PD1-Fc) as templates.
9
Generating HeLa Cell Line Expressing GFP-SEPT6
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HeLa (ATCC CCL‐2) or SEPT6‐GFP HeLa cells were cultured in DMEM (GIBCO) and 10% foetal bovine serum (FBS). The pLVX‐GFP‐SEPT6 lentiviral expression vector was created by inserting the human SEPT6_i3 (NP_665798) with a N‐terminal GFPtag into the pLVX‐puro vector (Takara Bio Inc.) 49 . Lentivirus was produced in HEK293FT cells by co‐transfection of pLVX‐GFP‐SEPT6 with psPAX2 and pMD2.g vectors at a ratio of 10:7:3 μg DNA, respectively (Trono laboratory second‐generation packaging system; Addgene). Supernatants from transfected HEK293FT cells containing the lentivirus were collected 24 and 48 h after transfection, pooled and filtered. HeLa cells were then infected with lentivirus for 48 h, and cells stably expressing GFP‐SEPT6 were selected by adding 1 μg/ml puromycin to the culturing media.
10
Overexpression of miR-34c in HUVECs
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For the overexpression of miR-34c, the human MIR34C flanking region was cloned into a lentiviral vector (pLVX-puro vector: Takara, Japan), as previously described (Kim et al., 2019 (link)). The pLVX-puro-miR-34c plasmids were cotransfected into HEK293T cells with psPAX2 (Gag-Pro-Plo) and pMD2.G (Env) using Lipofectamine 3000 (Invitrogen), according to the manufacturer’s instructions. The supernatants containing lentivirus were harvested and filtrated through a 0.45-μm syringe filter unit (Millipore, USA) after 48 and 72 h. One day before lentivirus infection, HUVECs were seeded into tissue culture plates. After polybrene (Millipore) was added to the virus supernatants at 8 μg/ml, the culture media was replaced with the virus particle-containing media. The second infection was performed after 24 h of incubation. One day after the second infection, the cells were changed to fresh EGM-2 media and treated with and without H2O2 at 700 nM for dysoxia. The cells were harvested after 24 h of incubation for further assays.
For hypoxia by gas-induction, HUVECs were transduced by lentiviruses containing MIR34C at one day after the cells were seeded and cultured in a hypoxic chamber (1% O2, 5% CO2, and 90% N2) for 72 h.
For hypoxia by gas-induction, HUVECs were transduced by lentiviruses containing MIR34C at one day after the cells were seeded and cultured in a hypoxic chamber (1% O2, 5% CO2, and 90% N2) for 72 h.
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