The elucidation of the mechanisms of neural development using glutamic acid decarboxylase knockout mice

1.利用谷氨酸脱羧酶(GAD)合成了γ-氨基丁酸(GABA)。我们分离并鉴定了小鼠GAD(mGAD-1和mGAD-2)基因。MGAD-1基因全长45kb，包含19个外显子，包括两个5‘非编码外显子(外显子0A和外显子0B)。另一方面，mGAD-2基因的长度超过50kb。我们构建了mGAD-2-靶向结构，用于产生基因敲除小鼠。将该载体电击入小鼠ES细胞，筛选出耐药克隆。我们选择了靶向干扰mGAD-2基因的克隆。这些克隆将被用于制造mGAD-2基因敲除小鼠，这些小鼠将有助于阐明mGAD基因在神经发育中的作用。2.为了了解mGAD-1基因表达调控的分子机制，我们分离了mGAD-1基因的5‘侧翼区。对5‘-侧翼区的序列分析表明，存在许多可能的调控元件，包括NRSE、KROX-24和Sp1。利用转基因小鼠，我们检测了10.2kb的mGAD-1基因片段(包括8.4kb的5‘序列)与细菌LacZ报告基因融合后的表达模式。在两个不同的创始人系中，转基因表达在特定脑区的神经元中，如嗅结节、大脑皮层和基底节，据报道GAD-1基因在这些区域表达丰富。这些结果表明，mGAD-1基因10.2kb的DNA片段可能是其在体内GABA能细胞特异性表达所必需的。

1.gamma-Aminobutyric acid (GABA) is synthesized by glutamic acid decarboxylase (GAD). We have isolated and characterized the mouse GAD (mGAD-1 and mGAD-2) genes. The mGAD-1 gene spans over 45 kb in length and contains 19 exons including two 5'noncoding exons (exon 0A and exon 0B). On the other hand, the mGAD-2 gene spans more than 50 kb in length. We made an mGAD-2-targetting construct for generating knockout mice. The construct was electroporated into mouse ES cells and drug-resistant clones were selected. We select the clones that have targeted disruption of the mGAD-2 gene. The clones will be used to make the mGAD-2 knockout mice and these mice would be useful to elucidate the role of mGAD genes in neural development.2.To understand the molecular mechanisms underlying the regulation of mGAD-1 gene expression, we have isolated the 5'-flanking region of the mGAD-1 gene. Sequence analysis of the 5'-flanking region revealed the presence of a number of putative regulatory elements including NRSE,Krox-24, and Sp1. Using transgenic mice, we have examined the expression pattern conferred by a 10.2 kb fragment of the mGAD-1 gene (including 8.4 kb of 5'sequences) fused to the bacterial lacZ reporter gene. In two separate founder lines, the transgene expression was observed in neurons of particular brain regions such as the olfactory tubercle, cerebral cortex, and basal ganglia, where expression of GAD-1 gene has been reported to be abundant. These results suggest that the 10.2-kb DNA fragment of the mGAD-1 gene may be essential for its GABA ergic cell-specific expression in vivo.