Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.
Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.
复制标题
转基因小鼠骨骼肌和心肌肌酸激酶基因调控元件分析。
DOI：
10.1128/mcb.16.4.1649
复制
发表时间：
1996
影响因子：
5.3
通讯作者：
Hauschka,SD
中科院分区：
文献类型：
--
作者：
Donoviel,DB;Shield,MA;Buskin,JN;Haugen,HS;Clegg,CH;Hauschka,SD
先前通过分析在培养的肌肉细胞中发现的小鼠肌肉肌酸激酶(MCK)基因的调节区，在转基因小鼠中进行了分析。在骨骼肌和心肌中高水平表达MCK-氯霉素乙酰转移酶融合基因需要位于NT-1256的206个碱基的MCK增强子；然而，与其在细胞培养中的行为不同，在增强子和基本启动子之间包含DNA的1-kb区域可以使骨骼肌的活性增加100倍。对增强子调控元件的分析也表明，它们在转基因肌肉和培养肌肉细胞中的性质存在重大差异。增强子Right E box或Carg元件突变的转基因表现出与野生型转基因没有区别的表达水平。MCK 1,256-BP 5‘区3个保守的E盒突变对大腿骨骼肌转基因表达也没有影响。所有这些突变都显著降低了培养的骨骼肌细胞的活性。然而，NT-1195上的富含AT增强子的元件对于转基因骨骼肌的表达是至关重要的。该位点的突变将骨骼肌的表达降低到与缺乏206-BP增强子的转基因相同的水平，尽管AT富含位点的突变不影响心肌的表达。这些结果表明，在培养的肌肉细胞和整个成年转基因肌肉中，MCK调节区的活性存在明显的差异。这表明，在不同的生理状态下，骨骼肌和心肌中存在着调控MCK基因的不同机制。
Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt –1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5′ region also had no effect on transgene expression in thigh skeletal muscle expression. All of these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt –1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscle cells and in whole adult transgenic muscle. This suggests that there are alternative mechanisms of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.