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

一种基因陷阱方法来识别参与中枢神经系统发育的新基因

• 批准号: 07557092
• 负责人: KUWANO Ryozo
• 金额: $ 13.7万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557092/
• 关键词: gene; cell abration; Cre-lox; diphteria toxin; gene trapping; toransgenic mice; brain; immunohistochemistry; 免疫組織科学; トランスジェニック; ネオマオシン耐性; マウス; 神経成長円錐; リコンビネース; cre-lox; 遺伝子発現; アストロサイト; 神経機能

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.

为了阐明参与神经元功能的神经元和神经胶质之间的相互作用，我们在转基因小鼠中使用白喉毒素a片段（DT-A）基因的细胞消融策略。基于星形胶质细胞中合适的启动子（如S100或GFAP启动子）和通过基因捕获发现的神经元中新启动子的时空特异性基因表达，DT-A基因在中枢神经系统的限制性细胞中被激活。此外，我们还介绍了cre-lox系统，用于靶基因的区域和阶段特异性调控。表达式。cre -重组酶介导的位点特异性重组发生在由34 bp（loxP）组成的限制性DNA序列上。在小鼠白血病启动子下游插入转录终止信号抑制了DT-A的活性。在创建转基因小鼠之前，必须在培养系统中验证cre-lox的细胞消融。我们证实了DT-A在转染细胞中的作用。我们制备了一些转基因小鼠系，其中一些由于位置效应而表现出异位表达模式。基因诱捕提供了一种在中枢神经系统和心脏中表达的新型启动子。重新构建了含有细胞消融绝缘子序列的靶基因。

项目成果

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T.K.Abe：“对从小鼠大脑中分离出的富含神经生长锥的部分中高度浓缩的膜蛋白进行二维分析和测序。”

Fujiwara, M.: "Expressions of a clacium - binding protein ( spot 35 / calbindin - D28k ) in mouse olfactory cells : possible relationship to neuronal differentiation." Eur. Arch. Otorhinolaryngol.254. 105-109 (1997)

Fujiwara, M.：“小鼠嗅细胞中钙结合蛋白（斑点 35/钙结合蛋白 - D28k）的表达：与神经元分化的可能关系。”

Shirai, M.et al.: "A gene trap strategy for identifying the gene expressed in the embryonic nervous system." Zool.Sci.13. 277-283 (1996)

Shirai, M.等人：“一种用于识别胚胎神经系统中表达的基因的基因陷阱策略。”

K.Ikeda: "Functional couplings of the delta‐and the kappa‐opioid receptors with the G‐protein‐activated K+channel." Biochem.Biophys.Res.Commun.208. 302-308 (1995)

K. Ikeda：“δ-和 κ-阿片受体与 G 蛋白激活的 K+ 通道的功能耦合。”Biochem.Biophys.Res.Commun.208 (1995)。

Fujiwara, M.et al.: "Expressions of a calcium-binding protein (spot35/calbindin-D28k) in mouse olfactory cells : possible relationship to neuronal differentiation." Eur.Arch Otorhinolaryngol. 254. 105-109 (1997)

Fujiwara, M.等人：“小鼠嗅细胞中钙结合蛋白（spot35/calbindin-D28k）的表达：与神经元分化的可能关系。”