E coli-based vectors for BAC delivery to mammalian cells

描述(由申请人提供)： 这项建议的总体目标是开发一种基于大肠杆菌的新型载体系统，用于修饰大片段的基因组DNA并将其功能性地输送到哺乳动物细胞中。覆盖人类基因组90%的大型BAC克隆的有序文库随时可用。这些BAC克隆包含完整的人类基因和周围的基因组序列，包括内源性启动子、内含子和顺式作用调控元件，这些调控元件将确保基因的准确时空表达。然而，使用这些有价值的BAC文库进行表达和功能研究一直受到修改如此大的DNA克隆和将它们送入哺乳动物细胞的困难。因此，我们建议在BAC宿主菌株DH10B中建立一种新的基于大肠杆菌的载体系统，该系统包括1)通过瞬时诱导表达大肠杆菌同源重组机制来方便地修饰BAC DNA；2)通过表达假结核杆菌侵袭素基因通过细菌入侵将DNA输送到哺乳动物细胞中。这一过程完全在宿主大肠杆菌中进行，不需要物理分离大的DNA分子，将有助于使用大的DNA构建物进行表达和功能研究。提出了该系统开发的三个具体目标。1)侵袭性大肠杆菌载体系统将被优化，用于在各种转化和原代培养的哺乳动物细胞中运送pGFP-neo报告质粒。2)将对人类基因组BAC进行修饰，并确定将其完整地输送到哺乳动物细胞进行表达研究的条件。3)人类人工染色体(HAC)的形成将通过构建改进的载体并将其受控地输送到哺乳动物细胞中来检测。这项拟议的研究将允许研究多种哺乳动物细胞中基因表达的组织和时间特异性。它将为基因表达提供包括HAC开发在内的新型基因传递载体，这将对开发治疗人类遗传性代谢性疾病和慢性病的基因治疗策略具有重要价值。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to develop a novel E. coli-based vector system for the modification of large pieces of genomic DNA and their functional delivery into mammalian cells. Ordered libraries of large BAC clones that cover >90% of the human genome are readily available. These BAC clones contain human genes complete with surrounding genomic sequences including endogenous promoters, introns, and cis-acting regulatory elements that will ensure accurate spatio-temporal gene expression. However, expression and functional studies using these valuable BAC libraries has been limited by difficulties in both modifying such large DNA clones and delivering them into mammalian cells. Therefore, we propose to develop a novel E. coli based vector system in BAC host strain DH10B that includes 1) convenient modification of BAC DNA by transient inducible expression of E. coli homologous recombination machinery and 2) delivery of DNA into mammalian cells via bacterial invasion by expression of the Y. pseudotuberculosis invasin gene. This process takes place entirely in the host E. coli without a requirement to physically isolate large DNA molecules and will facilitate the use of large DNA constructs for expression and functional studies. Three Specific Aims for the development of this system are proposed. 1) The invasive E. coli vector system will be optimized for delivery of pGFP-neo reporter plasmid in a variety of transformed and primary cultured mammalian cells. 2) Human genomic BACs will be modified and conditions determined for their intact delivery to mammalian cells for expression studies. 3) Human Artificial Chromosome (HAC) formation will be examined by construction of improved vectors and their controlled delivery into mammalian cells. This proposed research will permit the study of tissue and temporal specificity of gene expression in a wide variety of mammalian cells. It will provide novel gene delivery vectors, including HAC development, for gene expression, which will be valuable for development of gene therapy strategies for treatment of human genetic metabolic diseases and chronic diseases.