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）： 该提案的总体目的是开发一种新型的基于大肠杆菌的矢量系统，以修饰大型基因组DNA及其功能递送到哺乳动物细胞中。大型BAC克隆的有序文库覆盖了90％的人类基因组。这些BAC克隆包含人类基因，其中包含周围的基因组序列，包括内源性启动子，内含子和顺式作用调节元件，这些元件将确保准确的时空基因表达。但是，使用这些有价值的BAC文库的表达和功能研究受到修饰如此大的DNA克隆并将其输送到哺乳动物细胞中的困难受到限制。因此，我们建议在BAC宿主菌株DH10B中开发一种新型的基于大肠杆菌的媒介系统，其中包括1）通过Y. pseudotutuototutuototutuotototutuototototutuototototototototototobulsis Invasin Invasin Invasin Invasin Gene的表达，通过瞬时诱导大肠杆菌同源重组机制对BAC DNA进行方便修饰。这个过程完全发生在宿主大肠杆菌中，无需物理分离大的DNA分子，并将促进使用大型DNA构建体进行表达和功能研究。提出了该系统开发的三个特定目标。 1）将优化侵入性大肠杆菌载体系统，以在多种转化和原代培养的哺乳动物细胞中递送PGFP-NEO报告基因质粒。 2）将修改人类基因组BAC，并确定其完整递送至哺乳动物细胞进行表达研究的条件。 3）人造染色体（HAC）形成将通过构造改进的载体及其受控递送到哺乳动物细胞中检查。这项提出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新颖的基因递送载体，包括HAC发育，这对于制定基因治疗策略是有价值的，用于治疗人类遗传代谢疾病和慢性疾病。