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.

大脑中的神经元在从其增殖前体（尿路上皮干细胞）分化后立即退出细胞周期，并进入有丝分裂后状态，在该状态下，细胞在整个生命周期中保持不分裂。我们使用基因转移技术研究了分化神经元表现出的细胞生长永久停滞的机制。 Necdin 是一种 325 个氨基酸残基的蛋白质，定位于有丝分裂后神经元的细胞核，永久退出细胞周期。为了检查 necdin 是否赋予有丝分裂后表型，使用真核 lac 阻遏蛋白操纵子表达系统将 necdin cDNA 稳定转染到 NIH3T3 成纤维细胞中。当转染子被诱导表达异位necdin时，细胞生长被抑制，而细胞活力没有明显降低。为了阐明 necdin 基因表达的机制，我们分离并表征了小鼠 necdin 基因。 necdin 基因不含内含子，其上游区域缺乏典型的 TATA 和 CAAT 盒。使用转染荧光素酶报告基因的神经分化 P19 细胞对启动子区域进行删除分析，表明神经元限制性核心启动子位于 -80 至 -35 位，其中富含 G+C 结构域和假定的转录结合位点包含具有 PAS（per、arnt 和 single-thought）二聚化结构域的因子。这些结果表明，necdin 在神经上皮干细胞的神经元分化过程中诱导生长停滞，其中该基因的表达由位于 5' 上游区域的特定顺式作用元件介导。