A system for precise analysis of transcription-regulating elements of immunoglobulin genes.
BACKGROUND
Precise analysis of expression-regulating elements, such as enhancers and insulators, requires that they be tested under reproducible, isogenic conditions. The commonly used methods of transfecting DNA into cell lines and selecting for drug resistance lack the requisite precision, as they yield cell lines in which varying numbers of gene copies have inserted at varying and undefined sites. By contrast, recombination-mediated cassette exchange (RMCE), by which a site-specific recombinase is used to place a single copy of a transgene at a constant chromosomal site of a cell line, offers the necessary precision. Although RMCE is generally applicable, many regulatory elements of interest are tissue-specific in their function and so require cell lines in the appropriate ontogenetic state.


RESULTS
As reported here, we have used RMCE in a mouse B hybridoma cell line to establish a system with several additional advantages. To avoid the non-physiological features of prokaryotic DNA, this system uses the immunoglobulin mu heavy chain (IgH) gene from the hybridoma as the reporter. Expression can be measured simply by bulk culture assays (ELISA, Northern blot) and single cell assays (flow cytometry). Expression of the IgH reporter gene varies only 1.5 fold among independent transfectants, and expression is greatly (> 50 fold) increased by inclusion of the IgH intronic enhancer.


CONCLUSIONS
This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci.
Cheng
Emily Y
EY
Department of Immunology, University of Toronto, Toronto, Canada. emily.cheng@utoronto.ca
Collins
Cathy
C
Berru
Maribel
M
Shulman
Marc J
MJ
D000486:Allergy and Immunology; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D015152:Blotting, Northern; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004742:Enhancer Elements, Genetic; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005803:Genes, Immunoglobulin; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D006825:Hybridomas / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D007136:Immunoglobulins / Q000032:analysis* / Q000235:genetics*; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D014162:Transfection; D019076:Transgenes
D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007148:Immunoglobulin mu-Chains; D007136:Immunoglobulins; D004247:DNA