Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

Claire Robinson, Andreas Kolb

    Research output: Contribution to journalArticle

    8 Citations (Scopus)

    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 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. (C) 2008 Elsevier Inc. All rights reserved.

    Original languageEnglish
    Pages (from-to)508-522
    Number of pages15
    JournalExperimental Cell Research
    Volume315
    Issue number3
    DOIs
    Publication statusPublished - 1 Feb 2009

    Keywords

    • Mammary gland
    • Gene locus
    • Casein
    • Homologous recombination
    • Site-specific recombination
    • EMBRYONIC STEM-CELLS
    • DIRECTED GENOME MODIFICATION
    • CASEIN GENE
    • BETA-CASEIN
    • EXTRACELLULAR-MATRIX
    • GLUCOCORTICOID-RECEPTOR
    • TRANSCRIPTION
    • LINE
    • RECOMBINATION
    • SITE

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