E coli-based vectors for BAC delivery to mammalian cells

描述（由申请人提供）： 该方案的总体目标是开发一种新的E.基于大肠杆菌的载体系统，用于修饰大块基因组DNA并将其功能性递送到哺乳动物细胞中。覆盖>90%的人类基因组的大BAC克隆的有序文库是容易获得的。这些BAC克隆含有完整的人类基因，其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，这将确保精确的时空基因表达。然而，使用这些有价值的BAC文库的表达和功能研究受到修饰这样大的DNA克隆和将它们递送到哺乳动物细胞中的困难的限制。因此，我们建议开发一种新的E. coli载体系统，包括1）通过瞬时诱导表达E.大肠杆菌同源重组机制和2）通过表达Y. pseudotuberculosis invasin基因这个过程完全发生在主机E中。大肠杆菌中，而不需要物理分离大的DNA分子，并将促进使用大的DNA构建体的表达和功能研究。提出了开发该系统的三个具体目标。1)入侵E.大肠杆菌载体系统将被优化用于在各种转化的和原代培养的哺乳动物细胞中递送pGFP-neo报告质粒。2)将修饰人基因组BAC，并确定将其完整递送至哺乳动物细胞用于表达研究的条件。3)人类人工染色体（HAC）的形成将通过构建改进的载体及其受控递送到哺乳动物细胞中来检查。这项拟议的研究将允许在各种哺乳动物细胞中研究基因表达的组织和时间特异性。它将为基因表达提供新的基因递送载体，包括HAC的开发，这将对开发用于治疗人类遗传代谢疾病和慢性疾病的基因治疗策略具有价值。

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.