Dexamethasone responses in BRC-Abl (p210) transformed murine B-cells

For generation of pre-B cell lines, bone marrow from tibias and femurs of 4 to 8 week old C56Bl/6 mice (wt and vav-Gfi1b transgene) was infected with retroviral stocks of different BCR-Abl constructs (MSCV-GFP-IRESp210 or pSRa-p210-tkneo) and plated at a density of 5E+06 per 6 cm dish. All pre-B cells were fed twice a week and analyzed after they became stroma independent. Cells received 1E-08 M dexamethasone or vehicle for 12 hours prior to RNA extraction using TRIzol. mRNAs were converted into doublestranded cDNA using a T7-promoter tagged oligodT primer and transcribed by T7 RNA polymerase in the presence of biotinylated NTPs. Targets were hybridized to Affymetrix MOE430A arrays, washed, stained and scanned (GeneChip scanner 2500) as recommended by the manufacturer. Images were processed either by MAS5.0 (global scaling, TGT=1000) or RMAExpress (quantile normalization and background substraction) software tools.

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