Analysis of gene function using transgenic mice harboring antisense DNA.

本项目的目的是利用反义DNA构建降低靶基因表达的转基因小鼠，并根据这些转基因小鼠的表型揭示单个基因在体内的确切功能。最近，我们开发了髓鞘碱性蛋白（MBP）基因系统。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，因此出现了颤抖表型（Science 241, 593-595(1988)）。进一步的分析显示，MBP mRNA和MBP的减少发生在出生后两周左右，此时正常小鼠的MBP表达和髓鞘形成变得活跃。减少程度与颤抖表型的程度相关。此外，在反义MBP转基因中，各种与抗原性相关的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几种MBP mRNA的单一种类。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3个基因（β -actin、c-Ha-ras和dystrophin基因）的反义cDNA构建体，并产生了转基因小鼠。共建立了31个转基因系，但尚未发现明显的表型。目前所检测的几种转基因小鼠的反义基因表达量都很低。结合对这些转基因小鼠的广泛分析，我们现在正在改进引入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.