The Meq promoter of the virulent MDV 1 strain RB 1B was amplified by PCR with primers MEQ F and MEQ R. The promoter was first cloned into pCRW2. 1 TOPOW, then released by EcoRI digestion and re cloned into EcoRI linearized pd2EGFPCMV sellekchem producing the re porter plasmid pd2EGFP Meq. The chicken cDNA en coding the NFB p100 was released from the cloning vector pBS KS with HindIII and XbaI and inserted into HindIII and XbaI linearized expression vector pBK CMV, Inhibitors,Modulators,Libraries resulting in pBK CMV p100. The cDNA encoding the chicken NFB p105 cloned in pGEM4 was released by diges tion with EcoRI and KpnI and inserted Inhibitors,Modulators,Libraries into EcoRI and KpnI linearized pBK CMV, producing pBK CMV p105. The ankyrin repeats were removed from the 5 end of the NFB p105 cDNA by digestion with SacI.
The chicken Inhibitors,Modulators,Libraries NFB p65 cDNA cloned in pTZ18R was released by digestion with XhoI and MfeI and re cloned into XhoI and SmaI linearized Inhibitors,Modulators,Libraries pBK CMV producing pBK CMV p65. Plasmids were purified using the affinity chromatography columns and proper structure of all the plasmids was verified by restriction enzymes digest and sequencing. Promoter assays The activity of CD30 and Meq promoters was analyzed in vitro by promoter reporter assays. First, the reporter gene d2EGFP was placed under the control of the CD30 and Meq promoters and the coding sequences of tran scription factors were cloned into the expression plasmid pBK CMV. The promoter reporter plasmids and transcription factor ex pression plasmids were then transfected into SOgE cells, and the expression of the reporter gene was quan titatively measured by duplex real time PCR as described below.
SOgE cells Inhibitors,Modulators,Libraries were grown in Dulbeccos modified Eagles minimum essential medium supplemented with 10% fetal calf serum, penicillin, streptomycin and amphotericin B at 37 C with 5% CO2. Plasmids were transfected in triplicate into SOgE cells in 24 well plates at 80% confluence using LipofectinW reagent following the manufacturers instructions. Each well was transfected with 200 400 ng of DNA. To determine the effect of the Meq oncogene on the activity of the chicken CD30 promoters SOgE cells were transfected with either pUC18 alone, pd2EGFP N1 alone, pd2EGFP CD30 alone, or with a mix of pBK CMV Meq and pd2EGFP CD30. To determine the transactivation effect of the NFB transcription factors alone or in combin ation with the Meq oncoprotein on the Meq promoter SOgE cells were transfected with plasmid mixtures and DNA.
Plasmid pUC18 was added to transfection mixtures to give total amount of 400 ng plasmid DNA per well whenever it was necessary. Total RNA was isolated from transfected SOgE cells 48 h post transfection using TRI reagent following the manufacturers instructions. Isolated RNA was treated with DNaseI, extracted with phenol chloroform, precipitated with ethanol and resus pended not in water. The d2EGFP mRNA levels in transfected SOgE cells were quantified using the Platinum Quantitative RT PCR ThermoScript One Step System.