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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 the...

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Autores principales: Cheng, Emily Y, Collins, Cathy, Berru, Maribel, Shulman, Marc J
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2005
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1266055/
https://www.ncbi.nlm.nih.gov/pubmed/16202157
http://dx.doi.org/10.1186/1472-6750-5-27
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author Cheng, Emily Y
Collins, Cathy
Berru, Maribel
Shulman, Marc J
author_facet Cheng, Emily Y
Collins, Cathy
Berru, Maribel
Shulman, Marc J
author_sort Cheng, Emily Y
collection PubMed
description 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 μ 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. CONCLUSION: This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci.
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spelling pubmed-12660552005-10-26 A system for precise analysis of transcription-regulating elements of immunoglobulin genes Cheng, Emily Y Collins, Cathy Berru, Maribel Shulman, Marc J BMC Biotechnol Methodology Article 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 μ 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. CONCLUSION: This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci. BioMed Central 2005-10-04 /pmc/articles/PMC1266055/ /pubmed/16202157 http://dx.doi.org/10.1186/1472-6750-5-27 Text en Copyright © 2005 Cheng et al; licensee BioMed Central Ltd.
spellingShingle Methodology Article
Cheng, Emily Y
Collins, Cathy
Berru, Maribel
Shulman, Marc J
A system for precise analysis of transcription-regulating elements of immunoglobulin genes
title A system for precise analysis of transcription-regulating elements of immunoglobulin genes
title_full A system for precise analysis of transcription-regulating elements of immunoglobulin genes
title_fullStr A system for precise analysis of transcription-regulating elements of immunoglobulin genes
title_full_unstemmed A system for precise analysis of transcription-regulating elements of immunoglobulin genes
title_short A system for precise analysis of transcription-regulating elements of immunoglobulin genes
title_sort system for precise analysis of transcription-regulating elements of immunoglobulin genes
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1266055/
https://www.ncbi.nlm.nih.gov/pubmed/16202157
http://dx.doi.org/10.1186/1472-6750-5-27
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