E. COLI QUIESCENT SYSTEMS FOR MAMMALIAN EXPRESSION OF UNCLONABLE DNA

描述（由申请人提供）：所有表达基因的完整和准确收集对于任何基因组的基因组学和蛋白质组学研究至关重要。然而，尽管克隆所有表达的人类基因的重大努力，大约20%的预测的蛋白质编码基因是不完整的或没有发现在参考cDNA集合。大肠杆菌中大量蛋白质编码基因的不稳定性或毒性。大肠杆菌很可能在这个问题中起主要作用。目前研究的目标是开发线性和环状穿梭载体，将这些原本“不可克隆”的基因克隆到大肠杆菌中。大肠杆菌和哺乳动物细胞。载体在E.大肠杆菌，但它们将允许在哺乳动物细胞中活性表达。一个新的线性载体，显示高稳定性克隆，否则不稳定的DNA将被修改，以防止偶然表达在大肠杆菌。杆菌此外，它将与指导哺乳动物转录和翻译的信号偶联。该项目的一个主要好处是，它将使研究人员能够从人类和其他目前“不可克隆”的基因组中获得并表达各种基因。这些基因可能具有大的编码区（例如，>10 kb）、不寻常的碱基组成或重复序列。重要的是，这些载体将允许分析人类基因组的不稳定区域，这是许多遗传性疾病的原因。未来的衍生物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：表达基因的完整和准确的收集对于任何基因组的基因组学和蛋白质组学研究都是至关重要的。尽管在发现和克隆所有表达的人类基因方面付出了巨大努力，但大约20%的潜在蛋白质编码区是不完整的或在参考cDNA集合中未发现。不能将大量的蛋白质编码基因克隆到大肠杆菌中。大肠杆菌很可能在这个问题中起主要作用。目前研究计划的目标是开发线性和环状穿梭载体，以克隆在哺乳动物细胞中表达的其他“不可克隆”的基因。载体在E.大肠杆菌，但在哺乳动物细胞中有活性。一个新的线性载体，显示高稳定性克隆，否则不稳定的DNA将被修改，以防止偶然表达在大肠杆菌。杆菌此外，它将与指导哺乳动物转录和翻译的信号偶联。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的基因（例如，>10 kb）、不寻常的碱基组成或重复序列。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.