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 activation of B lymphocytes have contributed much to the understanding of the regulation of the B cell response to antigen. Primary, non-transformed B cells from the spleen in mice and the tonsils or peripheral blood 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 in culture. The influence of the transformed state on the results obtained using these models may limit their physiological relevance. This report describes a method whereby non-transformed B lymphocytes in primary culture can be transfected for use in studies of gene regulation in response to antigen receptor signals. Transfection was accomplished after only a 72 h exposure to LPS. The cells obtained after LPS treatment were greater than 97% pure, and more importantly, remained responsive to antigen-receptor generated signals. Responsiveness was confirmed by demonstrating induction of mRNA for the primary response gene egr-1, as well as induction of specific transcription factor binding activity in nuclear extracts from these cells. DEAE-dextran-mediated transient transfection was utilized to introduce an egr-1 promoter/reporter construct into these cells. This analysis of promoter activity yielded 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.
McMahon
S B
SB
Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia 19104.
Norvell
A
A
Levine
K J
KJ
Monroe
J G
JG
D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics; D051766:Early Growth Response Protein 1; D005786:Gene Expression Regulation; D017874:Immediate-Early Proteins; D007075:Immunoglobulin M / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation; D051379:Mice; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D011947:Receptors, Antigen, B-Cell / Q000502:physiology; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D007075:Immunoglobulin M; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011947:Receptors, Antigen, B-Cell; D014157:Transcription Factors