E. COLI QUIESCENT SYSTEMS FOR MAMMALIAN EXPRESSION OF UNCLONABLE DNA

描述(由申请人提供)：完整和准确地收集所有表达的基因对于任何基因组的基因组和蛋白质组研究都是至关重要的。然而，尽管克隆所有表达的人类基因付出了巨大的努力，但大约20%的预测蛋白编码基因是不完整的或在参考cDNA集合中找不到。在这个问题上，大肠杆菌中众多编码蛋白质的基因的不稳定性或毒性可能起到主要作用。目前这项研究计划的目标是开发线性和环形穿梭载体，将这些原本无法克隆的基因克隆到大肠杆菌和哺乳动物细胞中。这些载体将在大肠杆菌中转录沉默，但它们将允许在哺乳动物细胞中活跃表达。一种新的线性载体，对克隆其他不稳定的DNA表现出高度的稳定性，将被修改以防止在大肠杆菌中的偶然表达。此外，它还将与指导哺乳动物转录和翻译的信号相结合。该项目的一个主要好处是，它将允许研究人员从人类和其他基因组中获得并表达目前“无法克隆”的各种基因。这类基因可能具有较大的编码区(例如，10kb)、不寻常的碱基组成或重复序列。值得注意的是，这些载体将允许分析人类基因组的不稳定区域，这些区域是许多可遗传疾病的原因。未来的衍生品将包括专门用于细菌表达大的、有毒的或不稳定的cDNA的系统。公共卫生相关性：完整和准确的表达基因收集对于任何基因组的基因组和蛋白质组研究都是至关重要的。尽管人们努力发现和克隆所有表达的人类基因，但大约20%的潜在蛋白编码区是不完整的，或者在参考的cDNA库中找不到。无法将大量编码蛋白质的基因克隆到大肠杆菌中可能是这个问题的主要原因。目前这项研究计划的目标是开发线性和圆形穿梭载体，以克隆在哺乳动物细胞中表达的原本“不可克隆”的基因。这些载体在大肠杆菌中转录上是沉默的，但在哺乳动物细胞中是活跃的。一种新的线性载体，对克隆其他不稳定的DNA表现出高度的稳定性，将被修改以防止在大肠杆菌中的偶然表达。此外，它还将与指导哺乳动物转录和翻译的信号相结合。该项目的一个主要好处是，它将允许研究人员克隆具有大编码区(例如，10kb)、不寻常的碱基组成或重复序列的基因。未来的研究将包括开发类似的载体，专门用于原核表达大的或有毒的cDNA。

DESCRIPTION (provided by applicant): A complete and accurate collection of all expressed genes is critical for genomic and proteomic research of any genome. However, despite significant efforts to clone all the expressed human genes, approximately 20% of the predicted protein-coding genes are incomplete or not found in reference cDNA collections. The instability or toxicity of numerous protein-coding genes in E. coli is likely to play a major role in this problem. The goal of the current research proposal is to develop linear and circular shuttle vectors to clone these otherwise "unclonable" genes into E. coli and mammalian cells. The vectors will be transcriptionally silent in E. coli, but they will allow active expression in mammalian cells. A new linear vector that shows high stability for cloning otherwise unstable DNAs will be modified to prevent fortuitous expression in E. coli. Further, it will be coupled with signals to direct mammalian transcription and translation. A major benefit of this project is that it will allow researchers to obtain and express a wide variety of genes from the human and other genomes that are currently "unclonable". Such genes are likely to have large coding regions (e.g., >10 kb), unusual base composition, or repetitive sequences. Significantly, these vectors will allow analysis of unstable regions of the human genome, which are the cause of numerous heritable diseases. Future derivatives will include systems specifically for bacterial expression of large, toxic, or unstable cDNAs. PUBLIC HEALTH RELEVANCE: A complete and accurate collection of expressed genes is critical for genomic and proteomic research of any genome. Despite significant efforts to discover and clone all the expressed human genes, approximately 20% of the potential protein-coding regions are incomplete or not found in reference cDNA collections. The inability to clone numerous protein-coding genes into E. coli is likely to play a major role in this problem. The goal of the current research proposal is to develop linear and circular shuttle vectors to clone otherwise "unclonable" genes for expression in mammalian cells. The vectors will be transcriptionally silent in E. coli, but active in mammalian cells. A new linear vector that shows high stability for cloning otherwise unstable DNAs will be modified to prevent fortuitous expression in E. coli. Further, it will be coupled with signals to direct mammalian transcription and translation. A major benefit of this project is that it will allow researchers to clone genes that have large coding regions (e.g., >10 kb), unusual base composition, or repetitive sequences. Future research will include the development of similar vectors specifically for prokaryotic expression of large or toxic cDNAs.