Novel systems for mammalian expression, integration, and knockout of previously "

用于哺乳动物表达、整合和敲除先前“

• 批准号: 7910138
• 负责人: Ronald Godiska
• 金额: $ 60万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-10 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7910138
• 关键词: AT Rich Sequence; ATM gene; ATM wt Allele; Address; Alternative Splicing; Bacteria; Basic Science; Bypass; CMV promoter; Cell Culture Techniques; Cell Line; Cloning; Cloning Vectors; Complementary DNA; DNA; DNA Sequence; Defective Viruses; Development; Disease; Engineering; Enzymes; Escherichia coli; Eukaryotic Cell; Excision; Fragile X Syndrome; Friedreich Ataxia; Funding; G Protein-Coupled Receptor Genes; Generations; Genes; Genome; Goals; Guanine + Cytosine Composition; Human; Huntington Disease; In Vitro; Internal Ribosome Entry Site; Knock-out; Lead; Libraries; Mammalian Cell; Medical; Methodology; Methods; Molecular Biology; Muscular Dystrophies; Myotonic Dystrophy; Open Reading Frames; Organism; Phase; Physiology; Plasmids; Plasmodium; Pneumocystis carinii; Production; Proteins; Protocols documentation; Recombinant Proteins; Recovery; Refractory; Repetitive Sequence; Reporter Genes; Research; Research Proposals; Science; Shuttle Vectors; Site; Small Business Innovation Research Grant; Spinocerebellar Ataxias; Structure; System; T7 RNA polymerase; Technology; Testing; Transfection; Transgenic Animals; Transgenic Organisms; Trinucleotide Repeats; Universities; Variant; Vesicular stomatitis Indiana virus; Virus; Work; base; biosafety level 1 facility; clinically relevant; cost; drug discovery; expression cloning; expression vector; high throughput screening; improved; in vivo; innovation; mRNA capping; meetings; novel; promoter; protein expression; public health relevance; recombinase; success; tissue culture; tissue/cell culture; vaccine development; vector

DESCRIPTION (provided by applicant): The goal of this Phase II proposal is to develop systems for transient, stable, and transgenic expression of nearly any DNA sequence, particularly those that are impossible to clone into current vectors. Primary targets include genes containing long trinucleotide repeats, toxic ORFs, and very AT-rich regions of up to 50 kb. The systems will be based on a novel linear cloning vector. Variants will be developed for conventional transfection of eukaryotic cells and for generation of transgenic cell lines. In addition, we will incorporate the linear vector into a simple and robust method of transient expression, based on a modified Vesicular Stomatitis Virus ("VSV-T7"). Together these methods will allow mammalian expression and analysis of nearly any DNA sequence, in vivo or in vitro, including those that contain highly repetitive regions, have a high AT or GC content, or encode proteins that are toxic or deleterious to bacterial or mammalian hosts. This system will significantly expand the repertoire of genes that can be expressed and studied in mammalian cells, including cDNAs that have eluded cloning in high-throughput projects. Expression of otherwise toxic or unstable DNAs may lead to important advances in basic research and medical science. The systems described here will facilitate research on human Repeat Expansion Diseases, such as Fragile X Syndrome, Muscular Dystrophy, Friedreich ataxia, Huntington's Disease, Spinocerebellar ataxia, etc. They will allow cloning and expression of large eukaryotic genes, e.g., to analyze alternative splicing and co-express multiple proteins. They will also allow expression of proteins from pathogenic organisms, which often have very AT-rich genomes, such as Plasmodium sp., Pneumocystis carinii, and others. Importantly, the VSV-T7 system will simplify transient expression and make it amenable to high-throughput screening. PUBLIC HEALTH RELEVANCE: The proposed SBIR application, "Novel systems for mammalian expression, integration, and knockout of previously 'unclonable' genes", aims to provide a simple and efficient method to express high levels of recombinant proteins in mammalian cell cultures. This work will facilitate expression of numerous proteins that are currently refractory to cloning, and its ease of use and robust expression will enable high-throughput production of proteins in mammalian cells.

描述（由申请人提供）： 这个II期计划的目标是开发用于几乎任何DNA序列的瞬时、稳定和转基因表达的系统，特别是那些不可能克隆到当前载体中的DNA序列。主要目标包括含有长三核苷酸重复序列、毒性ORF和高达50 kb的AT丰富区域的基因。该系统将基于一种新的线性克隆载体。将开发用于真核细胞的常规转染和用于产生转基因细胞系的变体。此外，我们将基于经修饰的水泡性口炎病毒（“VSV-T7”）将线性载体并入瞬时表达的简单且稳健的方法中。这些方法一起将允许哺乳动物在体内或体外表达和分析几乎任何DNA序列，包括含有高度重复区域、具有高AT或GC含量或编码对细菌或哺乳动物宿主有毒或有害的蛋白质的那些。该系统将显著扩大可在哺乳动物细胞中表达和研究的基因库，包括在高通量项目中逃避克隆的cDNA。否则有毒或不稳定的DNA的表达可能导致基础研究和医学科学的重要进展。本文描述的系统将促进对人类重复扩增疾病的研究，例如脆性X综合征、肌营养不良、弗里德赖希共济失调、亨廷顿病、脊髓小脑共济失调等。来分析可变剪接和共表达多种蛋白质。它们还将允许表达来自病原生物体的蛋白质，这些病原生物体通常具有非常富含AT的基因组，例如疟原虫属，卡氏肺孢子虫等。重要的是，VSV-T7系统将简化瞬时表达，并使其适合高通量筛选。 公共卫生相关性： 拟议的SBIR申请，“哺乳动物表达，整合和敲除先前'不可克隆'基因的新系统”，旨在提供一种简单有效的方法，在哺乳动物细胞培养物中表达高水平的重组蛋白。这项工作将促进目前难以克隆的许多蛋白质的表达，其易用性和稳健的表达将使哺乳动物细胞中蛋白质的高通量生产成为可能。