Regulated expression of an insulin-responsive glucose transporter (GLUT4) minigene in 3T3-L1 adipocytes and transgenic mice.
Regulated expression of an insulin-responsive glucose transporter (GLUT4) minigene in 3T3-L1 adipocytes and transgenic mice.
复制标题
3T3-L1 脂肪细胞和转基因小鼠中胰岛素反应性葡萄糖转运蛋白 (GLUT4) 小基因的表达调节。
DOI：
10.1073/pnas.90.8.3348
复制
发表时间：
1993
影响因子：
11.1
通讯作者：
Lane,MD
中科院分区：
文献类型：
--
作者：
Ezaki,O;Flores-Riveros,JR;Kaestner,KH;Gearhart,J;Lane,MD
初步研究表明，胰岛素反应性葡萄糖转运蛋白 (GLUT4) 基因的 5' 侧翼序列长达 7 kb 不足以介导小鼠 3T3-L1 前脂肪细胞中分化诱导的报告基因表达。为了定位负责此功能的调控元件，构建了包含完整 GLUT4 基因（具有大量 5' 和 3' 侧翼序列）和一小段外源 DNA（用于转录鉴定）的小基因，并以相对较低的拷贝数转染到小鼠和 3T3-L1 前脂肪细胞中。在转基因小鼠中，GLUT4 小基因表现出与内源基因相似但不相同的组织特异性表达模式。在 3T3-L1 细胞中，小基因 mRNA 的表达在分化为脂肪细胞时发生，其动力学实际上与内源 GLUT4 mRNA 的动力学相同。在培养的脂肪细胞和转基因小鼠中，小基因的表达水平相对于内源基因而言较低。用 8-溴-cAMP 处理小基因转染的 3T3-L1 脂肪细胞，抑制内源 GLUT4 基因的转录，也抑制 GLUT4 小基因的表达。然而，下调内源性 GLUT4 基因转录的胰岛素未能正常下调 GLUT4 小基因的表达。这些发现表明，指导组织特异性表达（在心脏、骨骼肌和棕色脂肪组织中）、分化诱导的转录激活和 cAMP 诱导的转录抑制所需的顺式作用元件位于 14 kb GLUT4 小基因内。然而，最大组织特异性表达和胰岛素诱导的表达下调所需的顺式元件并不位于小基因中。
Preliminary studies showed that up to 7 kb of 5' flanking sequence of the insulin-responsive glucose transporter (GLUT4) gene are insufficient to mediate differentiation-induced reporter gene expression in mouse 3T3-L1 preadipocytes. To locate the regulatory element(s) responsible for this function, a minigene containing the entire GLUT4 gene with substantial 5' and 3' flanking sequence and a short segment of foreign DNA (for transcript identification) was constructed and transfected into mice and 3T3-L1 preadipocytes at relatively low copy number. In transgenic mice the GLUT4 minigene exhibited a pattern of tissue-specific expression similar, but not identical, to that of the endogenous gene. In 3T3-L1 cells expression of minigene mRNA occurred upon differentiation into adipocytes, with kinetics virtually identical to that of endogenous GLUT4 mRNA. In both cultured adipocytes and transgenic mice, the level of expression of the minigene was low relative to that of the endogenous gene. Treatment of minigene-transfected 3T3-L1 adipocytes with 8-bromo-cAMP, which represses transcription of the endogenous GLUT4 gene, also repressed expression of the GLUT4 minigene. However, insulin, which down-regulates transcription of the endogenous GLUT4 gene, failed to normally down-regulate expression of the GLUT4 minigene. These findings indicate that the cis-acting elements required for directing tissue-specific expression (in heart, skeletal muscle, and brown adipose tissue), differentiation-induced activation of transcription, and cAMP-induced repression of transcription are located within the 14-kb GLUT4 minigene. However, the cis elements necessary for maximal tissue-specific expression and for insulin-induced down-regulation of expression are not located in the minigene.