TRANSIENT TRANSFECTION OF MURINE B-LYMPHOCYTE BLASTS AS A METHOD FOR EXAMINING GENE-REGULATION IN PRIMARY B-CELLS

Studies of the biochemical and genetic processes associated with activ ation of B lymphocytes have contributed much to the understanding of t he regulation of the B cell response to antigen. Primary, non-transfor med B cells from the spleen in mice and the tonsils or peripheral bloo d in humans have proven to be informative models for dissection of the biochemical events leading to B cell activation. In contrast, genetic studies of this process have relied on transformed cell lines grown i n culture. The influence of the transformed state on the results obtai ned using these models may limit their physiological relevance. This r eport describes a method whereby non-transformed B lymphocytes in prim ary culture can be transfected for use in studies of gene regulation i n response to antigen receptor signals. Transfection was accomplished after only a 72 h exposure to LPS. The cells obtained after LPS treatm ent were greater than 97% pure, and more importantly, remained respons ive to antigen-receptor generated signals. Responsiveness was confirme d by demonstrating induction of mRNA for the primary response gene egr -1, as well as induction of specific transcription factor binding acti vity in nuclear extracts from these cells. DEAE-dextran-mediated trans ient transfection was utilized to introduce an egr-1 promoter/reporter construct into these cells. This analysis of promoter activity yielde d results which were indistinguishable from the pattern of expression of the endogenous egr-1 gene. Potential applications for dissection of transcriptional regulatory pathways in B lymphocytes are discussed.