PROGRAMMED KNOCKOUT MOUSE FOR THE ANALYSIS OF NEURAL ACTIVATORS.

为了研究与神经再生相关的分子的功能意义，我们设计了一种使用转基因策略的条件性功能丧失。为了实现这一目的，克隆了GAP-43启动子，并利用该启动子在神经损伤时表达反义mRNA或特定mRNA的核酶。以GAP-43/LacZ融合蛋白为报告基因检测启动子特异性。经过多次试验，获得了GAP-43启动子驱动表达的GAP-43/LacZ转基因。GAP-43/LacZ融合蛋白的表达模式与天然GAP-43非常相似。由于GAP-43的N端可以是朝向生长锥的靶向信号，因此在神经末梢中观察到LacZ染色。因此，这种转基因动物将是非常有用的可塑性和再生的进一步分析。此外，我们还建立了腺病毒介导的基因转移系统，这对于操纵某种基因的表达具有很大的优势。利用该腺病毒系统，探讨Ras信号通路在神经再生过程中的功能意义。目前，我们正在尝试使用我们已经制备的显性阴性Ras和Ras-GAP 1 m腺病毒在神经损伤后失去Ras的功能。使用这些腺病毒载体的分析正在进行中。

In order to investigate functional significance of molecules which are associated with nerve regeneration, we have designed a conditional loss of function using transgenic strategy. To achieve this, GAP-43 promoter was cloned, and this promoter was used to allow expression of antisense mRNA or ribozyme of certain mRNA when nerve got injury. GAP-43/LacZ fusion protein was used as a reporter to examine the promoter specificity. After several trials, GAP-43/LacZ transgene whose expression was driven by GAP-43 promoter was obtained. The expression pattern of GAP-43/LacZ fusion protein was very similar to that of native GAP-43. Since the N-terminus of GAP-43 can be a targeting signal toward growth cone, LacZ staining was observed in nerve terminal. Therefore this transgenic animal would be very useful for further analys of plasticity and regeneration. In addition, we have also established adenovirus mediated gene transfer system which could be a great advantage for the manipulation of a certain gene expression. Using this adenovirus system, a functional significance of Ras signaling pathway during nerve regeneration was accessed. At the moment, we are trying to lose function of Ras after nerve injury using dominant-negative Ras and Ras-GAP 1m adenovirus which we have already made. The analysis using these adenovirus vectors are now under going.