期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1990
卷号：87
期号：10
页码：3670-3674
DOI：10.1073/pnas.87.10.3670
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
