Palmiter R D, Sandgren E P, Koeller D M, Brinster R L
Department of Biochemistry, Howard Hughes Medical Institute, University of Washington SL-15, Seattle 98195.
Mol Cell Biol. 1993 Sep;13(9):5266-75. doi: 10.1128/mcb.13.9.5266-5275.1993.
DNA regions of 10 and 7 kb that flank the mouse metallothionein II (MT-II) and MT-I genes, respectively, were combined with a minimally marked MT-I (MT-I*) gene and tested in transgenic mice. This construct resulted in (i) position-independent expression of MT-I* mRNA and copy number-dependent expression, (ii) levels of hepatic MT-I mRNA per cell per transgene that were about half that derived from endogenous MT-I genes, (iii) appropriate regulation by metals and hormones, and (iv) tissue distribution of transgene mRNA that resembled that of endogenous MT-I mRNA. These features were not observed when MT-I* was tested without the flanking regions. These MT-I flanking sequences also improved the expression of rat growth hormone reporter genes, with or without introns, that were under the control of the MT-I promoter. Moreover, they enhanced expression from two of four heterologous promoters/enhancers that were tested. Deletion analysis indicated that regions known to have DNase I-hypersensitive sites were necessary but not sufficient for high-level expression. These data suggest that the DNA regions flanking the mouse MT-I and MT-II genes have functions like the locus control regions described for other genes.
分别位于小鼠金属硫蛋白II（MT-II）和MT-I基因两侧的10 kb和7 kb DNA区域，与一个标记最少的MT-I（MT-I*）基因相结合，并在转基因小鼠中进行测试。该构建体导致：（i）MT-I* mRNA的位置独立表达和拷贝数依赖性表达；（ii）每个转基因每个细胞中肝脏MT-I mRNA的水平约为内源性MT-I基因来源的一半；（iii）受金属和激素的适当调控；（iv）转基因mRNA的组织分布类似于内源性MT-I mRNA。当在没有侧翼区域的情况下测试MT-I*时，未观察到这些特征。这些MT-I侧翼序列还改善了受MT-I启动子控制的有或没有内含子的大鼠生长激素报告基因的表达。此外，它们增强了所测试的四个异源启动子/增强子中两个的表达。缺失分析表明，已知具有DNase I超敏位点的区域对于高水平表达是必要的，但不是充分的。这些数据表明，小鼠MT-I和MT-II基因两侧的DNA区域具有与其他基因所描述的基因座控制区域类似的功能。
