Studies on the Mechanisms of Neurogenesis using Gene Transfer Techniques

大脑中的神经元在从其增殖前体细胞(视网膜上皮干细胞)分化后立即退出细胞周期，并进入有丝分裂后状态，在此状态下细胞终生保持不分裂。我们利用基因转移技术研究了由分化的神经元显示的细胞生长永久停止的机制。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核内，永久退出细胞周期。为了验证Necdin是否具有有丝分裂后的表型，利用真核细胞Lac抑制因子-操纵子表达系统，将Necdin基因稳定地导入NIH3T3成纤维细胞。当转染体被诱导表达异位Necdin时，细胞生长被阻止，细胞存活率没有明显下降。为了阐明Necdin基因表达的机制，我们分离并鉴定了小鼠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。对转染荧光素酶报告基因的神经分化P19细胞的启动子区域的缺失分析表明，神经元限制性核心启动子位于-80到-35位，其中包含一个富含G+C的结构域和一个可能的转录因子与PAS(PER、ARNT和单一的)二聚体结构域的结合部位。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，该基因的表达是由位于5‘上游区域的特定顺式作用元件介导的。

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.