Whole genome transcription profile of antigen receptor activated B cells expressing wildtype or Calmodulin (CaM) - resistant E12

To elucidate the genome-wide role of Ca2+/ calmodulin inhibition of E2A in regulation of BCR activation, we performed DNA microarray analysis of activated splenic B cells expressing wildtype or CaM-resistant E12 mutant m8N47 with and without anti-IgM treatment for 3 hour.The expression of a remarkably large set of genes differed significantly. Primary splenic B-lymphocytes were purified from mice heterozygous for the deletion of E2A gene and infected with retroviruses expressing either wildtype or mutant E12 after activation with CD40L plus IL-4 for 14 h. After 12 h incubation, the infection was repeated for 12 h, followed by incubation for a further 22 h post-infection in fresh complete medium with the stimulants to allow expression of GFP and E12. In addition to CD40L plus IL-4, the medium was supplemented with LPS (200 ng/ml) during retroviral infection incubations to improve infection efficiency. To study gene expression following B-cell receptor activation, anti-mouse IgM (2.5 μg/ml) was added for 3 hour. For DNA microarray analysis of infected B-lymphocytes, the infected cells were purified with a FACSAria cell sorter instrument (BD Biosciences) using their green fluorescence from expression of the GFP.