Transfection of beta-casein chimeric gene and hormonal induction of its expression in primary murine mammary epithelial cells.
Transfection of beta-casein chimeric gene and hormonal induction of its expression in primary murine mammary epithelial cells.
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β-酪蛋白嵌合基因的转染及其在原代小鼠乳腺上皮细胞中表达的激素诱导。
DOI：
10.1073/pnas.87.10.3670
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发表时间：
1990
影响因子：
11.1
通讯作者：
T. Oka
中科院分区：
文献类型：
--
作者：
M. Yoshimura;T. Oka
为了研究酪蛋白基因表达的调控序列元件，我们构建了一个嵌合基因，该嵌合基因含有5.3 kb的小鼠β-酪蛋白基因的5 '-侧翼序列和1.6 kb的3'-侧翼序列，该嵌合基因与细菌氯霉素乙酰转移酶（CAT）基因融合。通过磷酸钙沉淀法将嵌合基因转染到从妊娠小鼠制备的原代小鼠乳腺上皮细胞中。转染程序对内源性β-酪蛋白基因的表达具有可忽略的影响。β-酪蛋白-CAT嵌合基因的表达需要胰岛素、氢化可的松和催乳素的协同作用。嵌合基因的表达也取决于适当的基质，因为嵌合基因的激素诱导的程度是高得多的细胞培养在重建的基底膜（基质胶）比细胞培养的I型胶原凝胶或塑料。另一方面，猴病毒40-CAT嵌合基因的表达，其中CAT基因由病毒的早期启动子驱动，不受激素环境的影响，并发生在塑料上培养的细胞中的最高水平。用一系列β-酪蛋白-CAT构建体进行的额外转染实验表明存在负责激素诱导的调控元件和负调控元件。
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.