Engineering stable, independent multi-transgene expression in mammalian cells

在哺乳动物细胞中构建稳定、独立的多转基因表达

• 批准号: 8320427
• 负责人: Andrew Steven Belmont
• 金额: $ 28.95万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8320427
• 关键词: Address; Area; Back; Bacterial Artificial Chromosomes; Basic Science; Biological Assay; Biological Products; Biology; Biomedical Research; Build-it; Cell Differentiation process; Cell Line; Cell Nucleus; Cell Proliferation; Cells; Chemicals; Chromatin; Chromatin Fiber; Chromatin Structure; Chromosome Positioning; Chromosomes; DHFR gene; DNA; Development; Disease; Engineering; Environment; Gene Amplification; Gene Expression; Gene Transfer Techniques; Generations; Genes; Genomics; Goals; Grant; Human; Interphase Chromosome; Investigation; Mammalian Cell; Marketing; Methodology; Methods; Mission; Molecular and Cellular Biology; Mus; NIH 3T3 Cells; Nucleic Acid Regulatory Sequences; Plague; Plasmids; Positioning Attribute; Procedures; Process; Processed Genes; Production; Property; Proteins; Regulatory Element; Reporter; Reporter Genes; Research; Site; Stem cells; Synthetic Genes; System; Technology; Testing; Tissue Engineering; Transcriptional Activation; Transfection; Transgenes; Viral; Viral Vector; Work; cell type; cellular engineering; embryonic stem cell; gene therapy; high throughput screening; improved; induced pluripotent stem cell; innovation; insight; novel; pluripotency; promoter; regenerative; tool; transdifferentiation; transgene expression

DESCRIPTION (provided by applicant): Achieving high level, reproducible, stable transgene expression in mammalian cells remains a major bottleneck to critical areas of biomedical research, including production of protein biopharmaceuticals, gene therapy, cellular reprogramming, tissue engineering, as well as basic research into fundamental molecular and cellular biology processes and mechanisms of disease. The rationale for the proposed research is to develop the optimal components for a single and multiple transgene BAC expression system that will overcome long- standing problems in mammalian transgene expression and find applications over a wide range of biomedical research areas. Our long-term goal is to overcome existing problems in mammalian transgene expression in order to achieve the ability to engineer entire synthetic gene networks into human cells for improved ex vivo gene therapy and tissue engineering applications. The specific aims of this proposal are to: 1. Identify appropriate DNA genomic regions, cloned within BACs, and promoters that can be used to drive copy number dependent, position independent expression of single and multiple transgenes. 2. Optimize BAC / promoter combinations through the minimization of BAC size and addition / deletion of appropriate cis regulatory regions. 3. Apply this technology to specific test "driver" applications requiring multi-gene expression, including improved methods for generating induced pluripotent stem cells and facilitated high-throughput screening for chemicals which modulate stem cell pluripotency and differentiation. We propose to develop a general methodology enabling the engineering in a single step any mammalian cell line to express stably any single protein, or set of multiple proteins, at levels comparable to 100s fold higher than endogenous genes. Completion of our proposal should result in an improved methodology for generation of iPS cells and transdifferentiation, and more broadly a new methodology for tissue engineering applications. Our approach is innovative because it builds on special insights derived from our basic science investigations into how 10 and 30 nm chromatin fibers fold into interphase chromosomes, and the relationship between this "large-scale chromatin structure" and transcriptional activation.

描述（由申请人提供）：在哺乳动物细胞中实现高水平、可重复、稳定的转基因表达仍然是生物医学研究关键领域的主要瓶颈，包括蛋白质生物药物的生产、基因治疗、细胞重编程、组织工程以及基础分子和细胞生物学过程和疾病机制的基础研究。所提出的研究的基本原理是开发用于单转基因和多转基因BAC表达系统的最佳组分，其将克服哺乳动物转基因表达中的长期存在的问题，并在广泛的生物医学研究领域中找到应用。 我们的长期目标是克服哺乳动物转基因表达中存在的问题，以实现将整个合成基因网络工程化到人类细胞中的能力，用于改进的离体基因治疗和组织工程应用。 这项建议的具体目标是：1.鉴定在BAC内克隆的适当DNA基因组区域，以及可用于驱动单个和多个转基因的拷贝数依赖性、位置非依赖性表达的启动子。2.通过最小化BAC大小和添加/删除适当的顺式调控区来优化BAC /启动子组合。3.将该技术应用于需要多基因表达的特定测试“驱动”应用，包括用于产生诱导多能干细胞的改进方法，以及促进调节干细胞多能性和分化的化学物质的高通量筛选。我们建议开发一种通用方法，使任何哺乳动物细胞系在一个单一的步骤工程化，以稳定表达任何单一的蛋白质，或一组多个蛋白质，在相当于100倍以上的水平比内源性基因。我们的建议的完成应导致一个改进的方法产生的iPS细胞和转分化，更广泛的组织工程应用的新方法。我们的方法是创新的，因为它建立在我们的基础科学研究中的特殊见解，即10和30 nm染色质纤维如何折叠成间期染色体，以及这种“大规模染色质结构”与转录激活之间的关系。