The ORF of ATP6V1A was cloned into pQCXIN (Addgene, USA) to generate packaged retroviruses in HEK293 cells following the manufacturer's instructions and by using TransIT-Lenti Transfection Reagent (Mirus bio, USA). Vero cells were transduced with packaged retroviruses and cultured in medium supplemented with puromycin for selection. The surviving cell clone was isolated, propagated, and examined for stable expression of ATP6V1A by Western blotting.
Transit lenti transfection reagent
Manufactured by Mirus Bio
Sourced in United States
The TransIT-Lenti Transfection Reagent is a laboratory product designed to facilitate the transfection of lentiviral vectors into target cells. It is a proprietary formulation of lipids and polymers that enables efficient delivery of genetic material into a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Lenti x concentrator, by Takara Bio (5 mentions)
Lenti x 293t cells, by Takara Bio (4 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Peg it virus precipitation solution, by System Biosciences (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Pspax2, by Addgene (3 mentions)
Polybrene, by Merck Group (3 mentions)
Ucoe sffv dcas9 bfp krab, by Addgene (2 mentions)
Sh800, by Sony (2 mentions)
8 well chamber slide, by Ibidi (2 mentions)
Mitotrackertm red cmxros, by Thermo Fisher Scientific (2 mentions)
Pmd2 g, by Addgene (2 mentions)
Puromycin, by Avantor (2 mentions)
Lentiviral transfer vector, by System Biosciences (2 mentions)
Dulbecco modified eagle medium (dmem), by Avantor (2 mentions)
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
Lentivirus concentration solution, by OriGene (2 mentions)
High bsa dmem, by Thermo Fisher Scientific (2 mentions)
Hek293ft cells, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
33 protocols using transit lenti transfection reagent
1
Stable ATP6V1A Overexpression in Vero Cells
2
Lentiviral Transduction for HMGB1 Expression
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The mouse HMGB1 gene was stably transduced to the knockout cells or wild-type cells using a Lentivirus system. Preparation of viral vector was as follows: 1 μg of a mouse HMGB1 lentiviral cDNA ORF clone, pLV-mHMGB1-GFPSpark (Sino Biological) and 1 μg 3rd Generation Packaging Mix (Applied Biological Materials) was transfected into 5 × 105 293 T cells (Takara Bio) using 6 μl TransIT-Lenti transfection reagent (Mirus Bio, LLC) at 37 °C for 48 h according to the manufacturer’s instruction. Recombinant virus was concentrated by Lenti-X Concentrator (Takara Bio), checked for its titer using the Lenti-X GoStix Plus (Takara Bio) and stored at − 80 °C until use. Lentiviral vector (3800 GoStix values compatible to 9.8 × 104 infection units) was transduced into 4 × 104 cells (MOI = 2.5) of knockout clones or WT cells with 8 μg/ml polybrene at 37 °C for 48 h. Then, the culture medium was changed to fresh medium not containing polybrene and the cells were diluted and put into a well of 96-well plate (0.2 cells/well) for single cell cloning. Expression of HMGB1-GFP fusion protein was confirmed by western blot analysis. After confirmation of HMGB1 expression, the cells were expanded and used for further study within two weeks of in vitro culture to avoid loss of HMGB1 expression.
3
Engineered Lentivirus for Modulating Hedgehog Pathway
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Lentiviral particles containing pMH0001 (UCOE-SFFV-dCas9-BFP-KRAB, Addgene #85969) were produced by transfecting HEK293T cells with standard packaging vectors using the TransIT-Lenti Transfection Reagent (Mirus Bio, MIR 6605). M10G cells were stably transduced with lentiviral particles to generate M10GdCas9-KRAB cells by isolating BFP-expressing cells using fluorescence activated cell sorting on a Sony SH800.
Single-guide RNA (sgRNA) protospacer sequences were individually cloned into the pCRISPRia-v2 vector (Addgene plasmid #84832), between the BstXI and BlpI sites, by ligation. Each vector was verified by Sanger sequencing of the protospacer. One sgRNA expression vector with the following protospacer was cloned for non-targeting control sgRNAs (ncRNA) (5′-GCTGCATGGGGCGCGAATCA-3′), PTCH1 sgRNAs (sgPTCH1) (5′-AAATGTACGAGCACTTCAAG-3′) and SMO sgRNAs (sgSMO) (5′-CAAGAACTACCGATACCGTG-3′). Lentivirus was generated as described above for each sgRNA expression vector, and M10GdCas9-KRAB cells were independently transduced with lentivirus from each sgRNA expression vector, then selected to purity using 20 μg/mL puromycin over 7 days.
Single-guide RNA (sgRNA) protospacer sequences were individually cloned into the pCRISPRia-v2 vector (Addgene plasmid #84832), between the BstXI and BlpI sites, by ligation. Each vector was verified by Sanger sequencing of the protospacer. One sgRNA expression vector with the following protospacer was cloned for non-targeting control sgRNAs (ncRNA) (5′-GCTGCATGGGGCGCGAATCA-3′), PTCH1 sgRNAs (sgPTCH1) (5′-AAATGTACGAGCACTTCAAG-3′) and SMO sgRNAs (sgSMO) (5′-CAAGAACTACCGATACCGTG-3′). Lentivirus was generated as described above for each sgRNA expression vector, and M10GdCas9-KRAB cells were independently transduced with lentivirus from each sgRNA expression vector, then selected to purity using 20 μg/mL puromycin over 7 days.
4
Lentiviral Particle Production in HEK293T Cells
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Lenti-X HEK293T cells (Takara Bio) were seeded at a density of 4.5 × 106 cells per 10 cm dish 18–24 h prior to transfection. The prepared cells were co-transfected using the TransIT®-Lenti transfection reagent (Mirus Bio) with MISSION® Genomics Lentivirus Packaging Mix (Mirus Bio) and lentiviral plasmids containing i53 variants/libraries of interest. The viral supernatant was collected 48 h after transfection, passed through a 0.45 μm filter (Cytiva), flash frozen, and stored until use at -80 °C. Viral titers were measured by FACS in K562 cells and were typically ~0.5–1.5 × 107 TU/mL.
5
Lentivirus Production and Transduction
6
Engineered Lentiviral System for ATRX, EZH2, and REST Modulation
7
Lentivirus Production for Cell Labeling
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Lentivirus production was performed as previously reported [11 (link)]. Briefly, lentiviruses were produced in Lenti-X 293T cells (Takara Bio, Shiga, Japan) by transfection of pMD2.G and psPAX2 with pLentiN-LAMP1-GFP or pLentiN-mCherry-LC3 using TransIT-Lenti Transfection Reagent (#MIR6600; Mirus, WI, USA). After 48 h, viral supernatants were filtered by a PVDF 0.45 μm filter (Millex-HV, Millipore, MA, USA).
8
Generating Lentiviral Vectors for AXL Protein Expression
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The pLVX-TetOne-Puro-hAXL WT and the mutant hAXL(c.G1353C, p.W451C) expression vectors were separately cotransfected into HEK-293T cells with the lentiviral packaging carrier plasmid pLP VSV-G (Nova Lifetech, Cat# PVT2326) and the lentiviral packaging plasmid psPAX2 (a gift from Didier Trono, Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID:Addgene_12260) using TransIT-Lenti Transfection Reagent (Mirus, Cat# MIR 6604) following the manufacturer’s recommended protocol. Virus-containing medium was harvested 48 hours after transfection, purified by passage through a 0.45 μm pore-size filter, and used to transduce recipient HEK-293T cells in the presence of 8 μg polybrene/mL (Sigma-Aldrich, Cat# TR-1003). The transduced cells were selected by 2 μg puromycin/mL. Induction of expression of WT and W451C-mutant AXL protein was achieved with 0.1 μg doxycycline/mL, applied 96 hours before harvesting cellular protein for further analyses. Negative control HEK-293T cells were transduced and selected, but received buffer instead of doxycycline.
9
Lentiviral Transduction for Bmi-1 Knockdown
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Briefly, a lentivirus vector (CS-H1-shRNA-EF-1α-EGFP) expressing short hairpin RNA (shRNA) against human Bmi-1 (target sequence CAGATGAAGATAAGAGAAT for sh-1; GAGAAGGAATGGTCCACTT for sh-2), and Luciferase was used (33 (link)), as described previously (25 ). HEK-293T cells, cultured in DMEM containing 10% heat-inactivated FBS and 1% Penicillin-Streptomycin, were cotransfected using TransIT Lenti Transfection Reagent (Mirus #MIR6600) with lentiviral packaging constructs (Gag-Pol and VSV-G Env). Virus was harvested and concentrated using Lenti-X Concentrator (Takara # 631232). A single lentiviral transduction was performed in culture dishes (Falcon 1008; Becton Dickinson) in the presence of Polybrene (8 µg/mL; Sigma #TR-1003-G).
10
Lentiviral Transduction of Ion Channels
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Viruses were produced as described previously.25 (link) For infections, 1 µg of constructs expressing human ion channel coding proteins RFP, Kv1.5-T2A-his-EGFP, or KCNJ2(Kir2.1)-Y242F-EGFP were used. For each construct, HEK293T cells at a confluency of 80% were used to transfect for virus production. Constructs were mixed with 0.5 µg helper (encoding gag/pol) and 0.1 µg encoding coat protein (pMD2) along with 6 µL TransIT-Lenti transfection reagent (MIR6603; Mirus Bio; Madison, WI) in 200 uL Opti-MEM (31,985,062; ThermoFisher Scientific; Waltham, MA). Culture media of HEK293T cells was replaced with Opti-MEM and 200µL of transfection mixture and cells were kept in transfection media overnight. Transfection media was replaced with DMEM containing 1% Pen/Strep the following day. Two days post-transfection media containing virus was collected and stored at −80 °C. Collection was repeated on the following day and the total virus collected was combined. To transduce cells, MDA-MB-231 or MDA-MB-468 cells were seeded in six-well plates at 2 × 105 cells per well in 2 mL DMEM containing 10 μg/mL polybrene and virus. The plates were centrifuged at 1500 r.p.m., 4 °C for 1 h and incubated at 37 °C. Medium was changed after 16 h. Infected cells were sorted by FACS and gating on eGFP-derived green fluorescence.
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