The hTERT-immortalised fibroblast cell lines ‘73–26' (Werner syndrome)24 (link) and wild-type sibling control (‘82–6')24 (link) were kindly provided by Judy Campisi (Buck Institute, CA), and the Werner syndrome cell line AG03141 (ref. 38 (link)) was kindly provided by David Kipling (Cardiff University). Retroviruses encoding wild-type or mutant human WRN protein were packaged in GP2–293 cells using the Retro-X Universal Packaging System (Clontech) according to the manufacturer's instructions. GP2–293 supernatants were used to transduce 73–26 hTERT cells in the presence of 4 μg ml−1 hexadimethrine bromide (Polybrene, Sigma) for 24 h. Following three successive rounds of transduction, cells were selected in 0.5 mg ml−1 G418 for 4–6 weeks. Resulting cultures were screened for WRN expression by western blotting using mouse anti-WRN (Abcam 66601, 1:500). All cell lines were tested and found to be mycoplasma-free before use.
Retro x universal packaging system
Manufactured by Takara Bio
Sourced in United States
The Retro-X Universal Packaging System is a lab equipment product designed for the storage and transportation of biological samples. It provides a standardized and secure packaging solution for a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Merck Group (4 mentions)
Rhs4346, by Horizon Discovery (2 mentions)
Ampho envelope vector, by Takara Bio (2 mentions)
Rela 1 trcn0000353629, by Merck Group (2 mentions)
Rela 2 trcn000029875, by Merck Group (2 mentions)
Rmm 4431 101264262, by Horizon Discovery (2 mentions)
Rmm 4431 200408572, by Horizon Discovery (2 mentions)
Hexadimethrine bromide polybrene, by Merck Group (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Retro x tet on advanced inducible expression system, by Takara Bio (1 mentions)
Lentiviral particles, by Merck Group (1 mentions)
Dmem f12 glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Retro x concentrator, by Takara Bio (1 mentions)
Insulin, by InvivoGen (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Retronectin, by Takara Bio (1 mentions)
Pretrox tet on advanced, by Takara Bio (1 mentions)
Puromycin, by Nacalai Tesque (1 mentions)
10 protocols using retro x universal packaging system
1
Immortalized Fibroblast Cell Lines for Werner Syndrome Research
2
Establishment of a Retroviral ERSE-Luciferase Reporter System
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Retroviral ERSE-luciferase vectors were used to produce recombinant retroviruses using standard methods. Briefly, pQCXIP-ERSE-Luciferase vectors were co-transfected with a VSV-G envelope on a separate plasmid (Clontech Retro-X Universal Packaging System, 631512) using lipofectamine and optiMem into the GP2-293 packaging cell line grown in antibiotic free, high glucose (4.5 g/L) DMEM supplemented with 1 mM sodium pyruvate, 10% fetal bovine serum and 4 mM L-glutamine. The resulting viral supernatant was harvested at 24 hr and 48 hr and used to transduce HEK293T (ATCC CRL-3216) cells that were then selected with puromycin. The stable cell line generated from the 2xERSE-luciferase construct (D9, PWM112) showed the best fold induction in response to ER stress and was used for the screen and all ERSE-luciferase assays in this manuscript. An early passage of 293T-D9 was expanded and frozen in aliquots such that the same passage of cells was used for each screening day.
3
Visualizing DNA Damage Response in A549 Cells
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A549 cells were transduced using a retroviral construct containing mCherry-53BP1-2 pLPC-Puro [Addgene plasmid # 19836; (15 (link))]. Retrovirus production and cells transduction with mCherry-53BP1 construct were carried out, as previously described (15 (link)). Retrovirus production was performed using Retro-X Universal Packaging System (Clontech), according to manufacturer’s instructions. Transduction was conducted by the incubation of cells and viral particles in a complete medium containing 8 μg/ml Polybrene (Sigma) at 37°C, 5% CO2. Selection of transduced cells was performed using 2 μg/ml of Puromycin (Gibco). A549 cells expressing mCherry-53BP1 were cultured in complete DMEM containing 0.4 μg/ml of Puromycin (Gibco). All cells were incubated at 37°C at 5% CO2 atmosphere. A549 cells expressing mCherry-53BP1 construct were counterstained for γ-H2AX marker as described (16 (link)). Fixed (4% paraformaldehyde, for 10 min) and permeabilized (0.1% Triton-X for 10 min) cells were labeled using primary anti-γ-H2AX antibody (1:100, Cell Biolabs) and secondary Alexa Fluor 488-conjugated donkey anti-mouse antibody (Molecular Probes).
4
Generation of MLL-AF9 Oncogenic Murine Model
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Bases 24–4208 encoding the residues 1–1395 of human KMT2A (Accession NM_001197104) and bases 1722–1994 encoding the residues 479–568 of human MLLT3 (Accession NM_004529) were amplified using PCR from human cDNA library and cloned into pMYs-IRES-Puro Retroviral Vector (pMYs-MLL-AF9-IRES-Puro). pMYs-MLL-AF9-IRES-Puro and pMYs-IRES-Neo Retroviral Vector, including Luciferase (pMYs-Luciferase-IRES-Neo), were packaged into viruses using the GP2-293 cell line (Retro-X™ Universal Packaging System, Clontech Laboratories, Inc.). c-Kit positive bone marrow cells were obtained from B6N (IGS): C57BL/6NCrl mice and infected with the packaged viruses, followed by clone selection using puromycin and neomycin. The cells were cultured with IMDM medium including 20% FBS, 10 ng/mL GM-CSF, 10 ng/mL IL-3, 10 ng/mL IL-6, 100 Units/mL penicillin, 20 ng/mL SCF, and 100 µg/mL streptomycin.
5
Generation of Chimera Gene-Inducible Cell Lines
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Chimera gene-inducible cell lines were generated by employing retroviral transduction using the Retro-X Tet-on Advanced Inducible Expression System and Retro-X Universal Packaging System (Clontech), basically as described previously [22] . In this system, Tet-on Advanced vector carrying the neomycin resistance gene and pRetroX Tight expression vector carrying the puromycin resistance gene were used. The 6 Â 10 5 Ba/F3 cells that introduced Tet-on Advanced maintained in the presence of neomycin were further infected with retrovirus of pRe-troX Tight including ATF7IP-PDGFRB in a total of 3 mL of medium and exposed for 10 hours. Cells were then washed, divided into two parts, and treated with puromycin for drug selection. One-half of the cells sequentially underwent drug selection. The subsequent stable resistant cells were cloned by repeated limiting dilution three times and used in the following experiments. The remaining half was further divided into two parts and treated with or without 1 mg/mL doxycycline (DOX) to induce the target proteins for 24 hours, and then IL-3independent cell proliferation was assessed with the cell proliferation assay. The Ba/F3 cells transduced with EBF1-PDGFRB, WT-PDGFRB, and empty vector, as mock, were similarly developed. Experiments were repeated at least three times independently, and representative data are presented.
6
Retroviral transduction of myeloma cells
7
Murine Fibroblasts and Myeloma Cells Manipulation
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Wild-type or gene-deficient C57BL/6 mice were housed at NII and used in accordance with the IAEC guidelines. MEFs, immortalized using 3T3 protocol, were generated from E13.5 embryos, as described.32 (link) HMCLs were a kind gift from Dr Michael Kuehl, NCI. Indicated transgenes were either constitutively expressed from MoMLV LTR promoter from pBabe.puro or inducibly expressed from an engineered promoter containing five tandem kappaB sites from HRS.puro retroviral construct. Retrovirus particle were generated from 293 T cells co-transfected with pCL-Eco.32 (link) For gene transduction into human-derived myeloma cells, pseudotyped retrovirus particles were produced using Retro-X Universal Packaging System (Clonetech, Mountain View, CA, USA) from GP2-293 cells co-transfected with pBabe.puro construct and Ampho envelope vector (Clonetech). Following shRNA-expressing lentiviral particles (Sigma Aldrich, St Louis, MO, USA) were used for knockdown in HMCLs: Rela#1-TRCN0000353629, Rela#2-TRCN000029875, Relb#1-TRCN0000280360, Relb#2-TRCN0000280361, Nfkb2#1-TRCN0000356047 and Nfkb2#2-TRCN0000356005, and control-SHC202V. For knockdown studies in fibroblasts, lentiviral constructs expressing control shRNA (#RHS4346) or RelB-shRNAs (RMM#4431-101264262 and RMM#4431-200408572) purchased from GE-Dharmacon (Lafayette, CO, USA) were used for generating lentiviral particles.49 (link)
8
Generating NTCP-Expressing Cell Lines
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To generate human NTCP-expressing cells, Daudi, HepG2, or Huh7 cells were transduced with a retroviral vector encoding NTCP or Flag tag-NTCP with 10 μg/mL of Polybrene (Sigma-Aldrich, St. Louis, MO). The retroviral vectors were generated by modification of a previously described method (31 (link)). Briefly, the retroviral expression plasmid and the envelope expression plasmid p10A1 were cotransfected into GP2-293 cells using the Retro-X universal packaging system (TaKaRa Bio Inc., Shiga, Japan). At 2 days posttransfection, the culture supernatants were filtered through 0.45-μm filters and then concentrated using a Retro-X concentrator (TaKaRa Bio USA). At 48 h postinfection, cells were subjected to an immunofluorescence assay or a viral infection assay. HepG2-NTCP and HepG2/Flag-NTCP cells were maintained in DMEM/F-12, GlutaMAX supplement (catalog no. 10565018; Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) in the presence of 50 μM hydrocortisone, 5 μg/mL of insulin, and puromycin (0.5 μg/mL; InvivoGen).
9
GLUT4 Overexpression in C57 Satellite Cells
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C57-SC were transduced with the pQCXIB retroviral plasmid containing the GLUT4 cDNA (59 , 60 (link)) using the Retro-X universal packaging system (Takara Bio, USA). SCs were seeded at a concentration of 25,000 cells per well in a 24-well plate; the following day, they were incubated with the viral particles for 24 hours. The transduction was either in the presence of polybrene (2 to 6 μg/ml) (Sigma-Aldrich) or on plates coated with RetroNectin (20 to 75 μg/ml) (Takara Bio, USA). The transduction medium was replaced after 24 hours, and the cells were incubated for 48 hours to allow expansion. Stable expressing cells were positively selected using blasticidin HCl antibiotic (10 μg/ml).
10
Establishing Inducible Cell Lines
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Platinum-E cells were transfected with pRetroX-Tet-On Advanced (Takara Bio), pRetroX-Tight-Pur-FLAG, pRetroX-Tight-Pur-FLAG-Srebf1(N), pRetroX-Tight-Pur-FLAG-Armc5, or pRetroX-Tight-Pur-FLAG-Armc5ΔBTB. Forty-eight hours after transfection, the media containing the ecotropic retroviruses were harvested, filtered, and transferred to 3T3-L1 cells using 10 μg/mL polybrene (Sigma-Aldrich). Infected cells were selected using 800 μg/mL G418 (Nacalai Tesque) and 2 μg/mL puromycin (Nacalai Tesque). The resultant cells were referred to as 3T3-L1–TetON-FLAG, 3T3-L1–TetON-FLAG-Srebf1(N), 3T3-L1–TetON-FLAG-Armc5, or 3T3-L1–TetON-FLAG-Armc5ΔBTB. Pantropic retrovirus were obtained using the Retro-X Universal Packaging system (Takara) with pRetroX-Tight-Pur-FLAG-hARMC5 or pRetroX-Tight-Pur-FLAG-hArmc5(R362L). These viruses were transferred to H295R cells without polybrene. Infected cells were selected using 800 μg/mL of G418 and 5 μg/mL of puromycin. The resultant cells were referred to as H295R-TetON-FLAG-hARMC5 or H295R-TetON-FLAG-hARMC5(R362L), respectively.
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