Physiological transgene regulation and functional complementation of a neurological disease gene deficiency in neurons.
Physiological transgene regulation and functional complementation of a neurological disease gene deficiency in neurons.
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神经元神经疾病基因缺陷的生理转基因调节和功能补充。
DOI：
10.1038/mt.2009.64
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Wade-Martins,Richard
中科院分区：
文献类型：
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作者：
Peruzzi,PierPaolo;Lawler,SeanE;Senior,SteveL;Dmitrieva,Nina;Edser,PaulineAH;Gianni,Davide;Chiocca,EAntonio;Wade-Martins,Richard
微管相关蛋白 tau (MAPT) 和 α-突触核蛋白 (SNCA) 基因在神经退行性疾病中发挥着核心作用。每个基因的突变都会导致家族性疾病，而两个基因座的常见遗传变异都会导致对散发性神经退行性疾病的易感性。在这里，我们使用基于单纯疱疹病毒 1 型 (HSV-1) 扩增子的感染性细菌人工染色体 (iBAC) 载体来表达大于 100 kb 的完整基因座，展示了人类 MAPT 和 SNCA 转基因位点的精细基因调控以及神经元细胞培养物和器官型脑切片中的功能互补。由携带 143 kbMAPT 或 135 kbSNCA 位点的 iBAC 载体转导的细胞培养物表达与内源基因相似的人类基因座。我们重点分析了携带完整MAPT基因座的iBAC-MAPT载体。在转导到神经元培养物中时，在严格的发育和细胞类型特异性控制下，从 iBAC-MAPT 表达多个 MAPT 转录本。通过酶联免疫吸附测定和免疫细胞化学检测，在 Mapt−/− 小鼠的原代神经元中，iBAC-MAPT 载体表达人 tau 蛋白，并恢复了离解神经元培养物和器官切片培养物中 Mapt−/− 神经元对 Aβ 肽处理的敏感性。该系统忠实保留基因表达和表型互补，为分析神经系统疾病基因提供了一种新方法。
The microtubule-associated protein tau (MAPT) and α-synuclein (SNCA) genes play central roles in neurodegenerative disorders. Mutations in each gene cause familial disease, whereas common genetic variation at both loci contributes to susceptibility to sporadic neurodegenerative disease. Here, we demonstrate exquisite gene regulation of the humanMAPTandSNCAtransgene loci and functional complementation in neuronal cell cultures and organotypic brain slices using the herpes simplex virus type 1 (HSV-1) amplicon-based infectious bacterial artificial chromosome (iBAC) vector to express complete loci >100 kb. Cell cultures transduced by iBAC vectors carrying a 143 kbMAPTor 135 kbSNCAlocus expressed the human loci similar to the endogenous gene. We focused on analysis of the iBAC-MAPTvector carrying the completeMAPTlocus. On transduction into neuronal cultures, multipleMAPTtranscripts were expressed from iBAC-MAPTunder strict developmental and cell type–specific control. In primary neurons fromMapt−/−mice, the iBAC-MAPTvector expressed the human tau protein, as detected by enzyme-linked immunosorbent assay and immunocytochemistry, and restored sensitivity ofMapt−/−neurons to Aβ peptide treatment in dissociated neuronal cultures and in organotypic slice cultures. The faithful retention of gene expression and phenotype complementation by the system provides a novel method to analyze neurological disease genes.