Analysis of gene function using transgenic mice harboring antisense DNA.

本项目的目的是构建利用反义DNA降低目的基因表达的转基因小鼠，并根据转基因小鼠的表型揭示单个基因在体内的确切功能。最近，我们建立了髓鞘碱性蛋白(MBP)基因的检测系统。在表达反义MBP RNA(与MBP mRNA互补的负链RNA)的转基因小鼠中，内源性MBP mRNA、MBP和中枢神经系统的髓鞘形成减少，从而出现更颤抖的表型(Science 241,593-595(1988))。进一步分析发现，正常小鼠出生后两周左右，MBP表达和髓鞘形成活跃时，MBP mRNA和MBP的表达减少。减少的程度与寒战表型的程度相关。此外，在反义MBP转基因中，所有与抗原性相关的MBPs同时减少，尽管使用的反义cDNA对应于通过选择性剪接产生的单一种类的MBP mRNA。为了将该系统应用于其他基因，我们在合适的启动子下构建了三个基因(β-肌动蛋白、c-Ha-ras和dystrophin基因)的反义cDNA结构，并获得了转基因小鼠。共建立了31个转基因株系，但尚未发现显著的表型。在目前检测到的几种转基因小鼠中，反义基因的表达很低，结合对这些转基因小鼠的广泛分析，我们现在正在改进导入的DNA的结构，以增加反义RNA的表达。

The aim of this project is to construct the transgenic mice which reduce the expression of the target gene using antisense DNA and to reveal the exact function of the individual gene in vivo based on the phenotype of these transgenic mice.Recently, we have developed this system for the myelin basic protein (MBP) gene. In the transgenic mice which expressed antisense MBP RNA (minus-strand RNA complimentary to a MBP mRNA), the endogeneous MBP mRNA, the MBP and the myelination in the central nervous system were reduced and consequently shiverer phenotype appeared (Science 241, 593-595 (1988)). Further analysis revealed that the reduction of MBP mRNA and MBP occurred around two weeks after birth when MBP expression and myelin formation of normal mice become active. The reduction level was correlated with the extent of shivering phenotype. Moreover, all kinds of the antigenically-related MBPs were simultaneously reduced in the antisense MBP transgenics, although the antisense cDNA used corresponded to the single kind of several MBP mRNA produced by alternative splicing.To apply this system for other genes, we have made the antisense cDNA constructs of three genes (beta-actin, c-Ha-ras and dystrophin genes) under the appropriate promoters and produced the transgenic mice. Total 31 transgenic lines were established but no prominent phenotype were recognized until now. The expression of the antisense transgene was very low in the several transgenic mice so far examined.Together with extensive analyses of these transgenic mice, we are now improving the structure of the introduced DNA to increase the antisense RNA expression.