Studies on the Mechanisms of Neurogenesis using Gene Transfer Techniques

利用基因转移技术研究神经发生机制

基本信息

批准号：
05454670
负责人：
YOSHIKAWA Kazuaki
金额：
$ 4.61万
依托单位：
Osaka University (1994-1995)Tokyo Metropolitan Institute for Neuroscience (1993)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

Neurons in the brain withdraw from the cell cycle immediately after differentiation from their proliferative precursors (reuroepithelial stem cells) , and enter the postmitotic state, in which the cells remain undivided throughout the lifetime. We have investigated the mechanisms underlying the permanent arrest of cell growth displayd by differentiated neurons using gene transfer techniques. Necdin is a 325 amino acid residue protein localized to the muclei of postmitotic neurons, which withdraw permanently form the cell cycle. To examine whether necdin confers the postmitotic phenotype, necdin cDNA was stably transfected into NIH3T3 fibroblasts using a eukaryotic lac repressor-operator expression system. When the transfectants were induced to express ectopic necdin, cell growth was arrested without appreciable reduction in cell viability. In order to elucidate the mechanisms underlying necdin gene expression, we have isolated and characterized the mouse necdin gene. The necdin gene contains no intron, and its upstream region lacks canonical TATA and CAAT boxes. Deletion analysis of the promoter region using neurally differentiated P19 cells transfected with luciferase reporter genes demonstrated that a neuron-restrictive core promoter is localized to positions -80 to -35, in which a G+C-rich domain and a putative binding site for transcription factors with PAS (per, arnt, and single-minded) dimerization domain are comprised. These results suggest that necdin induces growth arrest during neuronal differntiation of the neuroepithelial stem cells in which expression of this gene is mediated by the specific cis-acting elements located at the 5' upstream region.

大脑中的神经元与它们的增殖前体分化后立即从细胞周期中退出，并进入有丝分裂后状态，其中细胞在整个生命中保持不变。我们研究了使用基因转移技术通过分化神经元显示的细胞生长的永久性停滞的机制。 NECDIN是一种位于有丝分裂后神经元粘膜的325个氨基酸残基蛋白，该蛋白会永久撤回细胞周期。为了检查NECDIN是否赋予了有丝分裂后表型，使用真核lac抑制剂 - 操作器表达系统稳定地将Necdin cDNA稳定地转染到NIH3T3成纤维细胞中。当诱导转染剂表达异位坏死素时，会停止细胞生长，而不会明显降低细胞活力。为了阐明NECDIN基因表达的机制，我们已经分离并表征了小鼠Necdin基因。 NECDIN基因不含内含子，其上游区域缺乏规范的TATA和CAAT盒。使用神经分化的p19细胞对启动子区域进行删除分析，该p19细胞用荧光素酶报告基因转染表明，神经限制性的核心启动子位于-80至-35位置，其中G+C-富含C-富含C+C-C-富含C-C-C-C-C-富含型域和用于PAS转录因子的假定结合位点，用于PAS（每个，ARNT，ARNT，ARNT和单个界面）二聚体差异。这些结果表明，NECDIN在神经上皮干细胞的神经元不同过程中诱导生长停滞，其中该基因的表达是由位于上游5'上游区域的特定顺式作用元素介导的。