Romano Gaetano, Suon Sokreine, Jin Hao, Donaldson Angela E, Iacovitti Lorraine
Department of Neurosurgery, The Farber Institute for the Neurosciences, Thomas Jefferson University, Jefferson Hospital for the Neurosciences, Philadelphia, Pennsylvania, USA.
J Cell Physiol. 2005 Aug;204(2):666-77. doi: 10.1002/jcp.20319.
A DNA fragment of about 13 kb containing the human tyrosine hydroxylase (TH) promoter was previously isolated from a genomic DNA library and sequenced. The 11 kb from the transcription start of the human TH promoter was successively joined to the green fluorescent protein (GFP) to generate a transgenic mouse model. High levels of GFP expression could be observed in TH-positive cells of the Substantia nigra of embryonic and adult mice. Intriguingly, the sequence of the human TH promoter showed a low degree of homology with the mouse and rat TH promoters. In fact, comparative analysis of the sequences of human, rat, and mouse TH promoters revealed only five small regions of high homology. These five evolutionarily conserved regions were numbered in numeric progression from the 5' end of human TH promoter. In the present study, a panel of minimal human TH promoters was generated to analyze the transcriptional activity and specificity of gene expression conferred by the five conserved regions (CRs). The series of constructs was termed 250 bp and contained the first -194 bp of the human TH promoter immediately upstream of the transcription start, the first 35 bp the human TH messenger RNA leader, plus one or more of the five CRs. All the constructs were assembled in a self-inactivating form of the latest series of lentiviral vector system based on the human immunodeficiency virus type 1 (HIV-1). Lentiviral-mediated gene transfer was highly efficient for the in vitro transduction of human neuronal progenitor cells (hNPCs). Since a subset of hNPCs express TH following in vitro treatment with a mixture of differentiating agents, it was possible to assess specificity of expression for all the minimal human TH promoters. Overall, the successive addition of the five conserved regions produced a greater degree of specificity in induced TH-positive hNPCs, in particular after the addition of CRI (-8,917, -8,876). However, the human TH minimal promoters did not show any specificity for TH-positive differentiated mouse primary striatal and S. nigra cells, indicating a difference of TH gene regulation between the human and mouse systems. The human TH minimal promoters may provide the opportunity for the selection of TH-positive human embryonic and adult stem cells for brain transplantation experiments in animal models for Parkinson's disease.
先前从基因组DNA文库中分离并测序了一个约13 kb的包含人酪氨酸羟化酶（TH）启动子的DNA片段。将人TH启动子转录起始点上游11 kb的片段依次与绿色荧光蛋白（GFP）连接，构建了转基因小鼠模型。在胚胎和成年小鼠黑质的TH阳性细胞中可观察到高水平的GFP表达。有趣的是，人TH启动子序列与小鼠和大鼠的TH启动子显示出较低的同源性。事实上，对人、大鼠和小鼠TH启动子序列的比较分析仅发现了五个高度同源的小区域。这五个进化保守区域从人TH启动子的5'端开始按数字顺序编号。在本研究中，构建了一组最小化的人TH启动子，以分析这五个保守区域（CRs）赋予的转录活性和基因表达特异性。该系列构建体称为250 bp，包含转录起始点上游紧挨着的人TH启动子的前-194 bp、人TH信使RNA前导序列的前35 bp，以及五个CRs中的一个或多个。所有构建体均组装在基于1型人类免疫缺陷病毒（HIV-1）的最新系列慢病毒载体系统的自失活形式中。慢病毒介导的基因转移对人神经祖细胞（hNPCs）的体外转导效率很高。由于一部分hNPCs在用分化剂混合物进行体外处理后会表达TH，因此有可能评估所有最小化人TH启动子的表达特异性。总体而言，五个保守区域的依次添加在诱导的TH阳性hNPCs中产生了更高程度的特异性，特别是在添加CRI（-8,917，-8,876）之后。然而，人TH最小启动子对TH阳性分化的小鼠原代纹状体和黑质细胞没有显示出任何特异性，这表明人和小鼠系统中TH基因调控存在差异。人TH最小启动子可能为在帕金森病动物模型的脑移植实验中选择TH阳性的人胚胎和成人干细胞提供机会。
