Analysis of cerebellar function by using inducible mGluRl transgenic mice.

通过对小鼠特定脑区条件性表达mGluR 1的研究，探讨mGluR 1在神经元网络形成、突触、可塑性及学习记忆中的作用。为了获得这些小鼠，我们产生了mGluR 1转基因小鼠，其中mGluR 1的表达是由组织特异性启动子和四环素或强力霉素，使用转录因子tTA基因和tTA负责增强子，TRE。我们得到了以下结果。1. NSE-tTA Tg/lacZ-TRE-mGluR 1 Tg/mGluR 1（-/-）的产生和分析：该小鼠在小脑浦肯野细胞中表达mGluR 1，但Tg表达不能挽救在mGluR 1（-/-）小鼠中观察到的运动失调. CamKII-tTA Tg/lacZ-TRE-mGluR 1a双Tg的产生和分析：这些小鼠在3至4周龄时死亡。通过施用糊状食物，它们可以正常存活和生长。双Tg小鼠在嗅球、海马CA 1区和纹状体表达mGluR 1 mRNA，提示mGluR 1在这些区域的异位表达与Tg小鼠的致死性有关.在内源性mGluR 1位点表达tTA的小鼠的产生：由于mGluR 1的异位表达可能导致小鼠死亡，我们通过基因打靶将tTA基因导入mGluR 1位点。我们获得了mGluR 1（+/tTA）小鼠。此外，我们产生了11个独立的TRE-mGluRlTg. 4系。L7（+/tTA）敲入小鼠的产生：我们产生了在小脑浦肯野细胞中表达tTA的小鼠。我们还制备了表达TRE-PKCI或TRE-dominant negative MEK的Tgs的DNA构建体，其将分别抑制PKC途径或MAP激酶途径。

We planed to the role of mGluR1 in generation of neuronal network, synaptic, plasticity, and learning and memory by analysis of the mice, which conditionally express the mGluR1 in the specific brain region. In order to obtain these mice, we generated the mGluR1 transgenic mice in which expression of mGluR1 is regulated by tissue specific promoter and tetracycline or doxycylcine, using transcription factor tTA gene and tTA-responsible enhancer, TRE. We have obtained the following results.1. Generation and analysis of NSE-tTA Tg/lacZ-TRE-mGluR1 Tg/mGluR1 (-/-) : This mice express the mGluR1 in cerebellar Purkinje cells but Tg expression did not rescue the motor discoordination observed in mGluR1 (-/-) mice.2. Generation and analysis of CamKII-tTA Tg/lacZ-TRE-mGluR1a double Tg : These mice die at the ages of 3 to 4 weeks. They can survive and grow normally by application of paste food. The double Tg express mGluR1 mRNA in olfactory bulb, hippocampus CA1 area and striatum, suggesting the ectopic expression of mGluR1 in these regions is responsible for the lethality of the Tg mice.3. Generation of the mice that express tTA in endogenous mGluR1 locus : Since ectopic expression of mGluR1 might lead mice to death, we introduced the tTA gene into mGluR1 locus by gene targeting. We have obtained mGluR1 (+/tTA) mice. Further, we generated 11 independent lines of TRE-mGluR1 Tg.4. Generation of L7 (+/tTA) knock-in mice : We generated the mice that express tTA in cerebellar Purkinje cells. We also made DNA constructs for Tgs expressing TRE-PKCI or TRE-dominant negative MEK, which would inhibit PKC pathway or MAP kinase pathway, respectively.