A gene trap approach to identify a new gene involved in central nervous system development

在试图阐明神经元和神经胶质细胞之间的相互作用参与神经元的功能，我们使用了一种策略，细胞消融使用白喉毒素A片段（DT-A）基因在转基因小鼠。基于在星形胶质细胞中使用合适的启动子如S100或GFAP启动子的时空特异性基因表达以及通过基因捕获发现的新的神经元启动子，DT-A基因在中枢神经系统的限制性细胞中被激活。此外，我们还引入了cre-lox系统来对靶基因进行区域和阶段特异性调节。表情Cre重组酶介导的位点特异性重组发生在由34 bp组成的限制性DNA序列（loxP）上。DT-A活性被小鼠莫洛尼白血病病毒启动子下游转录终止信号的插入所抑制。在建立转基因小鼠之前，cre-lox的细胞消融必须在培养系统中进行验证。我们证实了DT-A在转染细胞中的作用。我们建立了几个转基因小鼠品系，其中一些表现出异位表达模式，由于位置效应。基因捕获提供了一个新的启动子表达在中枢神经系统和心脏。再次构建了含有绝缘子序列的用于细胞消融的靶基因的新构建体。

In attempt to elucidate of interaction between neuron and glia involved in neuronal function we used a strategy of cell ablation using a diphtheria toxin A fragment (DT-A) gene in transgenic mice. Based on spatial-temporal specific gene expression using an appropriate promoter such as S100 or GFAP promoter in astrocyte and a novel one for neuron discovered by gene trapping, the DT-A gene is activated in the restricted cells in the central nervous system. In addition we introduced cre-lox system for regional and stage specific regulation of the target gene. expression. Cre-recombinase mediated site specific recombination occurs on the restricted DNA sequence composed of 34 bp(loxP). DT-A activity was suppressed by insertion of transcription termination signal down stream of murine molony leukemia virus promoter. Cell ablation by cre-lox has to be verified in culture system prior to create transgenic mice. We confirmed an effect of DT-A in the transfected cells. We produced several transgenic mouse lines of which some showed ectopic expression pattern owing to a position effect.Gene trapping provided a novel promoter expressed in the central nervous system and heart. A new construct of the target gene containing insulator sequence for cell ablation is constructed again.