Transfection of beta-casein chimeric gene and hormonal induction of its expression in primary murine mammary epithelial cells.
To study the regulatory sequence elements responsible for casein gene expression, we constructed a chimeric gene containing 5.3 kilobases (kb) of the 5'-flanking sequence and 1.6 kb of the 3'-flanking sequence of the mouse beta-casein gene fused to the bacterial chloramphenicol acetyl-transferase (CAT) gene. The chimeric gene was transfected by the calcium phosphate-precipitation procedure into primary mouse mammary epithelial cells prepared from pregnant mice. The transfection procedure had negligible effect on expression of the endogenous beta-casein gene. Expression of the beta-casein-CAT chimeric gene required the synergistic actions of insulin, hydrocortisone, and prolactin. Expression of the chimeric gene also depended on the appropriate substratum because the degree of hormonal induction of the chimeric gene was much higher in cells cultured on a reconstituted basement membrane (Matrigel) than in cells cultured on either type I collagen gel or plastic. On the other hand, the expression of a simian virus 40-CAT chimeric gene in which the CAT gene was driven by the early promoter of the virus was not influenced by the hormonal milieu and occurred at the highest level in cells cultured on plastic. Additional transfection experiments with a series of beta-casein-CAT constructs suggested the existence of regulatory elements responsible for hormonal induction and negative regulatory elements.
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
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Yoshimura
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Oka
D000818:Animals; D002364:Caseins / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D002678:Chimera; D004357:Drug Synergism; D004848:Epithelium / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000187:drug effects*; D005796:Genes / Q000187:drug effects; D006854:Hydrocortisone / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011388:Prolactin / Q000494:pharmacology*; D015183:Restriction Mapping; D014162:Transfection
D002364:Caseins; D007328:Insulin; D011388:Prolactin; D006854:Hydrocortisone