Cloning and Characterization of the Promoter for the Mouse Insulin-Like Growth Factor Binding Protein-3 Gene

小白鼠類胰島素生長因子結合蛋白III基因啟動子之選殖及特性分析

劉宏仁(Hung-Jen Liu) ； Liam Murphy ； 廖明輝(Ming-Huei Liao) ； 張清棟(Ching-Dong Chang) ； 林茂勇(Maw-Yeong Lin)

《中華民國獸醫學會雜誌》 26卷3期 (2000/09) Pp. 167-174

https://doi.org/10.7009/JTCSVS.200009.0167

摘要

以人類的類膜島素生長因子結合蛋白III (insulin-like growth factor binding protein-3; IGFBP-3) 基因核酸探針來篩選小白鼠基因庫，成功篩選一段1. 3 kb的小白鼠IGFBP-3基因的啟動子(promoter)。並以限制酶、核酸序列及CAT (chloramphenicol acetyl transferase) 活性分析確認此基因片段(1.3 kb)。為確認此小白鼠IGFBP-3基因之啟動子是否具功能性，將1.3 kb 之小白鼠IGFBP-3 基因的啟動子及製備之缺損基因重組於pCAT-Basic載體，以磷酸鈣方法將這些重組載體轉染於大鼠初代肝細胞。經測試CAT的活性，證實選殖的1.3 kb 小白鼠IGFBP-3 基因的啟動子具有基礎的調控功能。以濃度10^(-8) M 之類胰島素生長因子I (insulin-like growth factor I; IGF- I)，生長荷爾蒙( growth hormone; GH) 及胰島素(insulin)等荷爾蒙，個別處理基因轉染的大鼠初代肝細胞，結果可分別提高CAT的活性3.2、1.9及1.5倍。CAT活性分析證實這些荷爾蒙可調控小白鼠IGFBP-3基因的啟動子。上述試驗證實具有調控功能之小白鼠IGFBP-3基因啟動子已成功被篩選及選殖。

關鍵字

類膜島素生長因子結合蛋白III基因 ； CAT報導因子 ； 大鼠初代肝細胞 ； 基因轉染作用 ； 啟動子

並列摘要

To clone the promoter for the mouse insulin-like growth factor binding protein-3 (IGFBP-3 ) gene, a mouse genomic library was screened using a human IGFBP-3 cDNA probe to obtain the 5'-flan king sequences. A 1.3-kb fragment containing the upstream sequence of the mouse IGFBP3 gene was obtained and partially analyzed. To determine whether the putative promoter of the 5'-flanking region of the mouse IGFBP-3 gene was functional, the 1.3-kb fragment of t he mouse IGFBP-3 gene was inserted upstream of the CAT reporter gene in t he pCAT-Basic vector. Rat collagenase prepared hepatocytes were transfected with the IGFBP-3 reporter gene construct, p1 .3CAT-S, using calcium phosphate. The 1.3-kb fragment gave a 5 folds higher activity than the pCAT-Basic vector, indicating this fragment contained basal promote r activity. Treatment of the transfected cells with IGF-I, GH, and insulin, increased the level of CAT activity by 3.2, 1.9, and 1.5 folds , respectively. IGF-I caused a high enhancement of promoter activity. GH and insulin were less effective than IGF-I in rat hepatocytes at a concentration of 10^(-8) M. The results revealed that the promoter for the mouse IGFBP-3 gene has been cloned and shown to be hormonally responsive.

並列關鍵字

Insulin-like growth factor binding protein-3 gene ； CAT reporter gene ； Rat primary hepatocytes ； Transfection ； Pormoter