The FH1-tUTG plasmid was a kind gift from A/Professor M. J. Herold (Walter and Eliza Hall Institute of Medical Research, Australia). The pcDNA3.1(+) and pEGFP-C1 plasmids were kind gifts from Professor Mian Wu (Translational Research Institute, Henan Provincial People's Hospital and People's Hospital of Zhengzhou University, Zhengzhou, P.R. China) and A/Professor Yongyan Wu (Department of Otolaryngology, Shanxi Key Laboratory of Otorhinolaryngology Head and Neck Cancer, the First Affiliated Hospital, Shanxi Medical University, Taiyuan, P.R. China), respectively. The pMDLg/pRRE plasmid (#12251, RRID: Addgene_12251), pMD2.g plasmid (#12259, RRID: Addgene_12259), and pRSV-Rev plasmid (#12253, RRID: Addgene_12253) were purchased from Addgene. The pEGFP-C1-eEF1α1-FL, pEGFP-C1-eEF1α1-D1, pEGFP-C1-eEF1α1-D2, pEGFP-C1-eEF1α1-D3, pEGFP-C1-eEF1α1-D1-Δ14-21, pEGFP-C1-eEF1α1-D1-Δ91-95, pEGFP-C1-eEF1α1-D1-Δ153-156, pcDNA3.1-MILIP, pcDNA3.1-MILIP-ΔE1, pcDNA3.1-MILIP-ΔE2, pcDNA3.1-MILIP-Δ-990/-1895, pcDNA3.1-MILIP-Δ-1488/-1895, pcDNA3.1-MILIP-R-FL, pcDNA3.1-MILIP-R-ΔE1, pcDNA3.1-MILIP-R-Δ-990/-1488, pcDNA3.1-MILIP-R-ΔDFOs, pcDNA3.1-MILIP-R1, and pcDNA3.1-MILIP-R2 plasmids were purchased from Sangon Biotech.
Pmd2 g plasmid
Manufactured by Addgene
Sourced in United States
The PMD2.G plasmid is a lentiviral packaging plasmid used for the production of lentiviral particles. It contains the envelope glycoprotein (VSV-G) that is required for the generation of pseudotyped lentiviral particles.
Automatically generated - may contain errors
Lab products found in correlation
Pspax2, by Addgene (15 mentions)
Pspax2 plasmid, by Addgene (14 mentions)
Polybrene, by Merck Group (11 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Pmdlg prre plasmid, by Addgene (4 mentions)
Prsv rev plasmid, by Addgene (3 mentions)
Jetprime transfection reagent, by Polyplus Transfection (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
Lenticrispr v2, by Addgene (2 mentions)
Lipod293 in vitro dna transfection reagent, by Addgene (2 mentions)
Plko 1 vector, by Merck Group (2 mentions)
Lego ig2, by Addgene (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hexadimethrine bromide, by Merck Group (2 mentions)
Jetprime, by Polyplus Transfection (2 mentions)
Polyethyleneimine, by Polysciences (2 mentions)
Puromycin, by Merck Group (2 mentions)
Pcmv dr8, by Addgene (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
42 protocols using pmd2 g plasmid
1
Plasmid Construction and Sources
2
Lentiviral Transduction of shRNA for NSUN5 Gene
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Four different sequence gene-specific short hairpin RNA molecules (shRNAs) for NSUN5 mRNA were designed and transduced into DBTRG-05MG and CAS-1 glioma cell line (for sequences please refer to supplementary Key Resources Table). shRNA against the MSS2 yeast mRNA (not present in mammals) was used as scrambled (control). All shRNA molecules were ligated into pLVX-shRNA2-ZsGreen plasmid (Clontech). 10 ug of each encoding plasmid was mixed with 7.5 μg of ps-PAX2 and 2.5 μg of PMD2.G plasmid (Addgene), using jetPRIME® Transfection Reagent (Polyplus Transfection). Upon 10 min of RT incubation, the transfection mix was added dropwise on a 10 cm culture plate containing HEK293-TLV lentiviral packaging cells at 80% confluence. After 72 h, medium with high-titer lentiviral particles was 0.45 μm filtered. DBTRG-05MG and CAS-1 glioma cell lines were cultured in virus containing medium for 24 h. After 5 passages, the green fluorescent cells were sorted by FACS and cultured in DMEM medium supplemented with 10% FBS and 1% v/v penicillin/streptomycin (Gibco).
3
Lentiviral Transduction of Rac1 in HEK293T
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HEK293T cells, grown 4 days in media without antibiotics and at 50–70% confluency, were co-transfected using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) with the pMD2.G plasmid, the psPAX2 plasmid (both from Addgene, Watertown, MA, USA) and the pLOC RAC1 wild-type plasmid at a 1:3:4 ratio, respectively. Six hours later, media was changed to media with 1M HEPES, 0.85%NaCl (Lonza, Houston, TX, USA) added to a final concentration of 25 mM, pH 7.2–7.5. The supernatant was collected in 1 mL aliquots 24, 48, and 72 h later and frozen at −80 °C. For transductions, polybrene (EMD Millipore, Burlington, MA, USA) was added to media containing 25 mM HEPES final as above, and the media mixture added 1:1 to thawed virus. Then, the final mixture was added to ~70% confluent KTC1 cells following manufacturer’s instructions. One day later, the media mixture with the virus was removed and replaced with fresh culture media. Cells were expanded in D-MEM/F12 culture media containing 10% FBS and 30 µg/mL blasticidin.
4
Lentiviral Transduction of PANC-1 Cells
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Lentivirus was generated in HEK293T cells (ATCC, Manassas, VA) in 225-cm2 flasks. Briefly, 22.2 μg of a CRISPR pooled gRNA library (human sgRNA library Brunello in lentiGuide-Puro) transfer vector (Addgene, cat. #73178) [17 (link)], 16.7 μg of psPAX2 plasmid (Addgene, cat. # 12260), and 11 μg of pMD2.G plasmid (Addgene, cat. # 12259) were combined with Lipofectamine 3000 (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol, and the mixture was used to transfect the cells. The virus-containing medium was collected 48 h after transfection and centrifuged at 500×g for 5 min to remove cells and debris. The supernatant containing the virus was then filtered with a 0.45-μM PES filter and frozen at − 80 °C. Viral transduction was accomplished by adding 150 μL of virus-containing medium per 1 × 106 cells (titer determined experimentally, MOI = 0.3) to 225-cm2 flasks of PANC-1 cells at 75% cellular confluence, along with 4 μg/mL polybrene (Sigma-Aldrich), for 16 h. The virus-containing medium was then replaced with fresh growth medium.
5
Plasmid Construction and Acquisition
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The FH1‐tUTG plasmid was a kind gift from Prof M. J. Herold (Walter and Eliza Hall Institute of Medical Research, Australia). The pMDLg/pRRE plasmid (#12 251), pMD2.g plasmid (#12 259), pRSU.pREV plasmid (#12 253), p27 cDNA (#20 420) and pCMVHA E2F1 (#24 225) were purchased from Addgene. The pGL3‐LIM27‐promoter and the pGL3‐LIMp27‐promoter‐ΔE2F1‐BR were constructed by Azenta Life Sciences (Suzhou, China). Other plasmids used in this study were generated by inserting the PCR products into the pcDNA3.1(+) or pcDNA3.1‐Flag vector by Azenta Life Sciences (Suzhou, China).
6
Generation of CRISPR-Cas9 Knockout Cell Lines
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We generated hHB−/−, RIG-I−/−, and MDA5−/− cells using a lentivirus-expressing CRISPR-Cas9 vector (lentiCRISPRv2; Addgene). The hHB-, RIG-I-, and MDA5-specific subgenomic RNA (sgRNA) sequences were the following: for hHB, 5′- GTA ACG GCA GAC TTC TCC TC-3′ (forward) and 5′-GAG GAG AAG TCT GCC GTT ACC-3′ ( reverse); for RIG-I, 5′-GGG TCT TCC GGA TAT AAT CC-3′ (forward) and 5′-GGA TTA TAT CCG GAA GAC CCC-3′ ( reverse); and for MDA5, 5′-CGA ATT CCC GAG TCC AAC CA-3′ (forward) and 5′-TGG TTG GAC TCG GGA ATT CGC-3′ ( reverse). Lenti-CRISPR virions were packaged in HEK293T cells by transfecting the psPAX2 plasmid (Addgene), the pMD2.G plasmid (Addgene), and either the lentiCRISPRv2 vector containing hHB-, RIG-I-, or MDA5-specific sgRNA or an empty lentiCRISPRv2 plasmid as a control. The suspensions were harvested at 72 h posttransfection (hpt). HEK293T cells were infected with the suspensions and treated with 1.5 μg/ml puromycin for 5 days. The cells were lysed, and hHB, RIG-I, or MDA5 expression was analyzed by Western blotting.
7
Lentiviral Vector Production in HEK293T Cells
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In 100 mm dishes, HEK293T cells were transfected using 6 μg of the proposed plasmid, 4 μg of psPAX2 (Addgene plasmid, # 12260) and 2 μg of pMD2.G plasmid (Addgene plasmid, #12259) per well with LipoD293™ In Vitro DNA Transfection Reagent. The transfected cells were incubated under normal conditions for 48 h and then harvested. The supernatant of HEK293T cell culture was collected after centrifugation at room temperature (RT). Concentration of virus was quantified using Lentivirus Concentration Solution (Genomeditech, #GM-040801-100) following manufacture's protocol. The lentivirus was stored at −80°C before use.
8
Lentiviral Knockdown of TRIT1 in DMS-273 Cells
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Lentiviral plasmids for TRIT1 human shRNA (TL300819-C, Origene, Rockville, MD, USA) and scrambled shRNA (TR30021, Origene, Rockville, MD, USA), both cloned in pGFP-C-shLenti vector, were used. To obtain the lentiviral particles, 10 μg of plasmid were mixed with 7.5 μg of ps-PAX2 and 2.5 μg of PMD2.G plasmid (Addgene, Watertown, MA, USA), using jetPRIME® Transfection Reagent (Polyplus Transfection, New York, NY, USA). The transfection mix was added to HEK293 cells at 80% confluence. After 72 h, medium with high-titer lentiviral particles was 0.45 μm-filtered and DMS-273 cells were cultured in virus containing medium for 24 h. After five passages, green fluorescent cells were sorted by fluorescence-activated single cell sorting (FACS).
9
Lentiviral Vector Production in HEK293T Cells
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HEK293T cells were cultured in individual 15 cm dishes for each viral construction. When the cells reached approximately 70–80% confluency (~ 1.5 × 107 cells), they were cotransfected with 15 μg of the pLVX-LAT2 plasmid, 15 μg of the psPAX2 plasmid (Addgene #12260, Cambridge, MA, USA) and 10 μg of the pMD2.G plasmid (Addgene #12259, Cambridge, MA, USA) plasmid with Lipofectamine 2000 transfection reagent. At 18 h post-transfection, the medium was replaced with 25 mL of fresh complete medium. The supernatants were harvested at 48 h and 72 h, centrifuged at 1000 rpm for 10 min at 4 °C to remove the cells, and filtered through a 0.45 μm filter to remove the debris. Finally, the supernatant was ultracentrifuged at 120,000×g for 2 h at 4 °C, dissolved in PBS after the removal of the supernatant, and stored at − 80 °C.
10
CRISPR Knockout of Cell Signaling Genes
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SgRNA sequences targeting Cdk4, Cdk6, Sting, Mavs (primer sequences are listed in Supplementary Table 1 ) were designed and synthesized, then cloned into LentiCRISPR V2 vector plasmid (Addgene, #52961). Lentivirus was obtained from HEK293T cells by co-transfection of psPAX2 plasmid (Addgene, #12260), pMD2.G plasmid (Addgene, #12259) and Cdk4-sgRNA plasmid, Cdk6-sgRNA, Mavs-sgRNA plasmid, Sting-sgRNA plasmid or control vector plasmid with Lipofectamine 3000 (Thermo Fisher Scientific) according to the provider’s protocols for 48 hours. After passing through a 0.22 μm filter, lentivirus supernatant was collected and then stored at −80 °C. The lentivirus stocks were used to infect TC-1 cells or MCA205 cells. After 48 h infection, cells were cultured in selective medium containing 4ug/ml puromycin (InvivoGen, #ant-pr-1) for at least one week. Monoclonal cells were obtained from FACSAria™ III cell sorter (Becton Dickinson, San José, CA, USA) and cultured in 96-well plate. The knock-out cell lines were identified by Western Blot assay.
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