Germline transcription and recombination of a murine VDJmudeltagamma1 transgene.
To investigate the regulation of Ig switch recombination, we have constructed mice with a 56 kb VDJmudeltagamma1 transgene. This transgene included an anti-nitrophenyl VDJ segment, Smu, Cmu, Cdelta, Igamma1, Sgamma1, Cgamma1 and the Cgamma1 membrane exons from the murine Igh(a) haplotype. Two founder lines were produced, with very similar characteristics. Transgenic B cells expressed normal amounts of Cmu (which is >95% transgenic), Cdelta and other cell surface markers, and normal amounts of VDJ and Cmu RNA. Gamma1 germline transcription of the transgenes is properly regulated since stable transcripts were not expressed in B cells treated with lipopolysaccharide (LPS) alone, nor in thymus or non-lymphoid tissues, but were expressed after treatment of B cells with LPS + IL-4 or CD40L + IL-4. B cells from both lines of transgenic mice expressed transgenic gamma1a after in vitro culture with CD40L + IL-4, but not after culture with CD40L alone. However, the CD40L + IL-4 induced IgG1 precursor frequency is much lower for VDJmudeltagamma1 transgenic B cells (1:240-760) than for non-transgenic B cells (1:9). Analysis of DNA from transgenic hybridomas indicated that switch recombination can take place in switch (S) regions, but can also take place outside S regions. These results indicate that targeting of switch recombinase to S regions must include regulation beyond the S regions themselves and correct germline transcription. This additional regulation might include cis-acting elements or appropriate spacing or arrangement of the recombining elements.
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109-0620, USA.
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Cunningham
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Ackerly
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Claflin
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J
Collins
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Wu
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Ford
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R
Lansford
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Alt
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Dunnick
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D023201:CD40 Ligand; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics*; D005803:Genes, Immunoglobulin / Q000235:genetics*; D006241:Haptens / Q000276:immunology; D006825:Hybridomas; D017578:Immunoglobulin Class Switching / Q000235:genetics*; D007133:Immunoglobulin Joining Region / Q000235:genetics; D007134:Immunoglobulin Switch Region / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007138:Immunoglobulin delta-Chains / Q000032:analysis / Q000235:genetics; D007142:Immunoglobulin gamma-Chains / Q000032:analysis / Q000097:blood / Q000235:genetics; D007148:Immunoglobulin mu-Chains / Q000032:analysis / Q000235:genetics; D015847:Interleukin-4 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D019076:Transgenes / Q000235:genetics*
D006241:Haptens; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D007138:Immunoglobulin delta-Chains; D007142:Immunoglobulin gamma-Chains; D007148:Immunoglobulin mu-Chains; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D023201:CD40 Ligand; D015847:Interleukin-4