Far upstream regulatory elements enhance position-independent and uterus-specific expression of the murine alpha1(I) collagen promoter in transgenic mice.
Far upstream regulatory elements enhance position-independent and uterus-specific expression of the murine alpha1(I) collagen promoter in transgenic mice.
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发表时间：
1999
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通讯作者：
K. Krempen;Doris Grotkopp;Keith Hall;Alexandra J. Bache;Andrea Gillan;R. Rippe;D. Brenner;M. Breindl
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作者：
K. Krempen;Doris Grotkopp;Keith Hall;Alexandra J. Bache;Andrea Gillan;R. Rippe;D. Brenner;M. Breindl
许多真核基因的阶段和组织特异性表达受顺式调节元件的调节，其中一些元件位于转录起始位点附近，而另一些元件已在相当远的距离处被鉴定。在以前的研究中，我们已经确定了远在上游的DNase I-超敏感位点的鼠α 1（I）胶原蛋白（Col 1a 1）基因，这可能发挥了调节这个丰富的表达基因的作用。在这里，我们已经克隆了几个这些网站到报告基因构建体含有Col 1a 1启动子驱动的绿色荧光蛋白（GFP）报告基因，并测试其可能的功能，在转染实验和转基因小鼠。在瞬时和稳定转染中，没有一个超敏位点对Col 1a 1启动子活性有显著影响，表明它们不包含经典的转录增强子。在转基因动物中，位于-18至-19.5 kb的一个元件增强了连接的Col 1a 1启动子的位置非依赖性活性，并且可能是基因座控制区的一部分。另一个位于-7至-8 kb的元件特异性增强转基因小鼠子宫中报告基因的表达，表明它含有一种新的转录增强子，该增强子可能参与调节动情周期期间子宫组织重塑中I型胶原表达。我们的研究还证明了GFP报告基因用于转基因动物的多功能性，因为它可以在活动物，整个安装胚胎，组织学薄切片，或原代细胞培养物中进行分析，它可以在组织或细胞提取物中使用荧光计非常灵敏地定量。
The stage- and tissue-specific expression of many eukaryotic genes is regulated by cis-regulatory elements, some of which are located in proximity to the start site of transcription whereas others have been identified at considerable distances. In previous studies we have identified far upstream DNase I-hypersensitive sites in the murine alpha1(I) collagen (Col1a1) gene, which may play a role in the regulation of this abundantly expressed gene. Here we have cloned several of these sites into reporter gene constructs containing the Col1a1 promoter driving the green fluorescent protein (GFP) reporter gene and tested their possible functions in transfection experiments and transgenic mice. In transient and stable transfections none of the hypersensitive sites had a significant effect on Col1a1 promoter activity, indicating that they do not contain a classical transcriptional enhancer. In transgenic animals one element located at -18 to -19.5 kb enhanced the position-independent activity of the linked Col1a1 promoter and may be part of a locus control region. Another element located at -7 to -8 kb specifically enhanced reporter gene expression in the uteri of transgenic mice, suggesting that it contains a novel transcriptional enhancer that may be involved in the regulation of type I collagen expression in tissue remodeling in the uterus during the estrous cycle. Our studies also demonstrate the versatility of the GFP reporter gene for use in transgenic animals because it can be analyzed in live animals, whole mount embryos, histological thin sections, or primary cell cultures, and it can be quantified very sensitively in tissue or cell extracts using a fluorometer.