Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion
Abstract
The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A {beta}-galactosidase reporter gene was inserted in place of the {beta}-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the {beta}-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal {beta}-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when insertedmore »
- Authors:
-
- Molecular Recognition Group, Hannah Research Institute, Ayr KA6 5HL (United Kingdom)
- Publication Date:
- OSTI Identifier:
- 21176174
- Resource Type:
- Journal Article
- Journal Name:
- Experimental Cell Research
- Additional Journal Information:
- Journal Volume: 315; Journal Issue: 3; Other Information: DOI: 10.1016/j.yexcr.2008.10.029; PII: S0014-4827(08)00431-X; Copyright (c) 2008 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0014-4827
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 60 APPLIED LIFE SCIENCES; CASEIN; CHROMATIN; GALACTOSIDASE; GENE REGULATION; GENES; HORMONES; MAMMARY GLANDS; MICE; RECOMBINATION; STEM CELLS; THYMIDINE; TRANSCRIPTION
Citation Formats
Robinson, Claire, and Kolb, Andreas F. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion. United States: N. p., 2009.
Web. doi:10.1016/j.yexcr.2008.10.029.
Robinson, Claire, & Kolb, Andreas F. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion. United States. https://doi.org/10.1016/j.yexcr.2008.10.029
Robinson, Claire, and Kolb, Andreas F. 2009.
"Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion". United States. https://doi.org/10.1016/j.yexcr.2008.10.029.
@article{osti_21176174,
title = {Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion},
author = {Robinson, Claire and Kolb, Andreas F.},
abstractNote = {The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A {beta}-galactosidase reporter gene was inserted in place of the {beta}-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the {beta}-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal {beta}-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the {beta}-casein gene.},
doi = {10.1016/j.yexcr.2008.10.029},
url = {https://www.osti.gov/biblio/21176174},
journal = {Experimental Cell Research},
issn = {0014-4827},
number = 3,
volume = 315,
place = {United States},
year = {Sun Feb 01 00:00:00 EST 2009},
month = {Sun Feb 01 00:00:00 EST 2009}
}