pcpgl-basic  (GenScript corporation)

Journal: Journal of Virology

Article Title: Restricted TET2 Expression in Germinal Center Type B Cells Promotes Stringent Epstein-Barr Virus Latency

doi: 10.1128/JVI.01987-16

Figure Lengend Snippet: TET2 restores EBNA2 activation of the methylated LMP1 promoter. The ability of EBNA2 to activate the unmethylated, methylated, and 5hmC-modified forms of the EBV LMP1 promoter was analyzed using reporter gene assays. LMP1p was cloned upstream of the luciferase gene in the CpG-free vector pCpGL-basic and was methylated (dark gray) or mock treated (light gray) in vitro. The construct was transfected into EBV− BJAB cells with and without TET2, which converts 5mC into 5hmC (black), in the presence and absence of EBNA2. Luciferase assays were performed 2 days posttransfection. The fold luciferase activation is shown relative to the activity of the unmethylated vector transfected with control vectors, which is set to 1. Error bars indicate standard errors calculated from 2 replicate experiments consisting of triplicate conditions.

Article Snippet: The EBV LMP1 ED-L1 promoter inserted into the pCpGL-basic reporter gene construct was methylated or mock treated in vitro using CpG methyltransferase M.SssI (New England BioLabs [NEB]) according the manufacturer's instructions.

Techniques: Activation Assay, Methylation, Modification, Clone Assay, Luciferase, Plasmid Preparation, In Vitro, Construct, Transfection, Activity Assay