E coli-based vectors for BAC delivery to mammalian cells

用于将 BAC 递送至哺乳动物细胞的大肠杆菌载体

• 批准号: 7013563
• 负责人: PETER E WARBURTON
• 金额: $ 28.83万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7013563
• 关键词: DNA binding protein; Escherichia coli; Yersinia; artificial chromosomes; bacterial genetics; biotechnology; cell line; gene delivery system; gene expression; gene targeting; gene therapy; human genetic material tag; molecular cloning; plasmids; reporter genes; tissue /cell culture; transfection; transfection /expression vector

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.

描述（由申请人提供）： 该提案的总体目标是开发一种新型的基于大肠杆菌的载体系统，用于修饰大片段基因组 DNA 并将其功能性递送到哺乳动物细胞中。覆盖超过 90% 的人类基因组的大型 BAC 克隆的有序文库很容易获得。这些 BAC 克隆包含完整的人类基因以及周围的基因组序列，包括内源启动子、内含子和顺式作用调节元件，以确保准确的时空基因表达。然而，使用这些有价值的 BAC 文库进行表达和功能研究受到了修饰如此大的 DNA 克隆并将其递送到哺乳动物细胞中的困难的限制。因此，我们建议在 BAC 宿主菌株 DH10B 中开发一种新的基于大肠杆菌的载体系统，包括 1) 通过大肠杆菌同源重组机制的瞬时诱导表达来方便地修饰 BAC DNA，以及 2) 通过表达假结核杆菌侵袭素基因，通过细菌入侵将 DNA 递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，无需物理分离大 DNA 分子，并将有助于使用大 DNA 构建体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) 侵入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递送 pGFP-neo 报告质粒。 2) 将修改人类基因组 BAC，并确定将其完整递送至哺乳动物细胞进行表达研究的条件。 3) 将通过构建改进的载体并将其控制递送至哺乳动物细胞来检查人类人工染色体（HAC）的形成。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。它将提供用于基因表达的新型基因递送载体，包括HAC开发，这对于开发治疗人类遗传代谢疾病和慢性疾病的基因治疗策略具有重要价值。