Engineering stable, independent multi-transgene expression in mammalian cells

描述(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，包括生产蛋白质生物制药、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研究。这项研究的基本原理是开发一个单一和多个转基因BAC表达系统的最佳成分，该系统将克服哺乳动物转基因表达的长期问题，并在广泛的生物医学研究领域找到应用。我们的长期目标是克服哺乳动物转基因表达方面存在的问题，以便能够将整个合成基因网络工程到人类细胞中，用于改进体外基因治疗和组织工程应用。这一建议的具体目的是：1.确定在BAC中克隆的合适的DNA基因组区域和启动子，这些启动子可以用来驱动单个和多个转基因的位置无关的表达。2.通过最小化BAC大小和添加/缺失适当的顺式调控区域来优化BAC/启动子组合。3.将这项技术应用于需要多基因表达的特定“驱动”应用，包括改进的产生诱导多能干细胞的方法，以及促进高通量筛选调节干细胞多能性和分化的化学物质。我们建议开发一种通用的方法，使任何哺乳动物细胞系在一步工程中就能稳定表达任何单一蛋白质或多个蛋白质组，水平相当于内源基因的100倍。我们的提案的完成将导致iPS细胞生成和转分化的改进方法，更广泛地说，将为组织工程应用提供新的方法。我们的方法是创新的，因为它建立在我们的基础科学研究的特殊见解的基础上，这些研究涉及10 nm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。

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.