Mouse Trp53 transcript variant 1 (NM_011640.3) was cloned into the retroviral vector pMSCV-IRES-GFP II (Addgene plasmid #52107). Trp53 point mutations were introduced using site-directed mutagenesis with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs E0552S). Retrovirus was produced by plasmid transfection into Platinum-E (Plat-E) cells. Lentivirus to express Cre recombinase was produced by transfecting NIH HEK293T cells with Cre-IRES-PuroR plasmid (Addgene plasmid #30205), Δ8.9 and VSV-G. All transfections were performed with FuGENE 6 transfection agent (Promega E2691) in Opti-MEM Reduced Serum Medium (Gibco 31985070) for 5 hours, and the media replaced with standard DMEM/F12, 10% FBS for viral production. Virus was collected 48, 72, and 96 hours post transfection and combined.
Fugene 6 transfection agent
Manufactured by Promega
Sourced in United States
FuGENE 6 is a non-liposomal, proprietary transfection reagent. It is designed to facilitate the delivery of DNA, RNA, and other compounds into a variety of mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Pmscv ires gfp 2, by Addgene (2 mentions)
Cre ires puror plasmid, by Addgene (2 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (2 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (2 mentions)
Genistein, by Merck Group (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
Daidzein, by Merck Group (1 mentions)
Qpcr reagents, by Bio-Rad (1 mentions)
Dual luciferase reporter dlr assay system, by Promega (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Adipored assay reagent, by Lonza (1 mentions)
Britelite plus, by PerkinElmer (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Corning (1 mentions)
Celltiter glo, by Promega (1 mentions)
Platinum e retroviral packaging cells, by Cell Biolabs (1 mentions)
5 protocols using fugene 6 transfection agent
1
Retroviral Overexpression of Trp53 Mutants
2
Adipogenic Differentiation Assay
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Quantitative polymerase chain reaction (qPCR) reagents were obtained from BIO-RAD. Rosiglitazone, genistein, and daidzein were purchased from Sigma. The -specific antagonist GlaxoWellcome9662 (GW9662) was a gift from GlaxoSmithKline Pharmaceuticals. AdipoRed assay reagent was bought from Lonza, and Hoechst 33342 from Sigma. Adipocyte differentiation kits (Adipogenesis Assay Kit item no. 10006908) were purchased from Cayman Chemical Company. Fugene 6 transfection agent and the Dual-Luciferase® Reporter (DLR™) assay system were purchased from Promega. qPCR oligos were obtained from Invitrogen.
3
Influenza A Luciferase Reporter Assay
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293 T cells in Dulbecco’s modified Eagle’s medium (DMEM) minus phenol red, supplemented with 10% heat inactivated FBS, 1% sodium pyruvate and 1% L-glutamine (Cellgro, Manassas, VA) were transfected with pCAGGS expression vector encoding A/H3N2/Udorn/72 PB2, PB1, PA, NP and Influenza A Luciferase reporter plasmid with a 1:1:1:1:0.5 PB2:PB1:PA:NP with Fugene 6 transfection agent (Promega, Madison, WI) in OptiMEM (Gibco, Carlsbad, CA). Transfected cells were treated with compound 2 hours post-transfection, and plates were incubated for 48 hours at 37 C, 5%CO2. Compounds were prepared at 10 mM in 100% DMSO and added as a 1:3 8-point serial dilution with a 50 μM final concentration. Following incubation, cells were lysed and luciferase production quantified with the addition of Britelite Plus (Perkin-Elmer, Waltham, MA), with a 1:1 transfection mix:Britelite Plus ratio. CellTiter-Glo (Promega, Madison, WI) was added to cytotoxicity plates following manufacturer’s instructions to quantify cell survival.
4
Retroviral Transduction of JAK2 Variants
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Murine JAK2WT (WT) and murine JAK2V617F cDNA15 (link) were cloned into the retroviral vector pMCs-IRES-GFP (Cell Biolabs, San Diego, CA, USA). Transient transfection of Platinum-E retroviral packaging cells (Cell Biolabs) was performed by using the FuGENE6 transfection agent (Promega, Madison, WI, USA) according to the manufacturer's protocol. Retroviral supernatants were harvested after 48 h and used to transduce the murine interleukin (IL)-3 -dependent pro-B cell line Ba/F3 or bone marrow cells.
5
Retroviral Expression of Mutant Trp53
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Mouse Trp53 transcript variant 1 (NM_011640.3) was cloned into the retroviral vector pMSCV-IRES-GFP II (Addgene plasmid #52107). Trp53 point mutations were introduced using site-directed mutagenesis with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs E0552S). Retrovirus was produced by plasmid transfection into Platinum-E (Plat-E) cells. Lentivirus to express Cre recombinase was produced by transfecting NIH HEK293T cells with Cre-IRES-PuroR plasmid (Addgene plasmid #30205), Δ8.9 and VSV-G. All transfections were performed with FuGENE 6 transfection agent (Promega E2691) in Opti-MEM Reduced Serum Medium (Gibco 31985070) for 5 hours, and the media replaced with standard DMEM/F12, 10% FBS for viral production. Virus was collected 48, 72, and 96 hours posttransfection and combined.
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