Simultaneous Control of Expression of Two Genes in Mammalian Tissue Culture

同时控制哺乳动物组织培养中两个基因的表达

• 批准号: 7321976
• 负责人: Claude Maina
• 金额: $ 13.22万
• 依托单位: NEW ENGLAND BIOLABS, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-20 至 2008-08-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7321976
• 关键词: Agonist; Attention; Binding; Biochemical Pathway; Cell Line; Cell Therapy; Cells; Chromatin; Cloning; Cloning Vectors; Code; Communities; Complement; Condition; DNA; DNA Binding Domain; DNA biosynthesis; Development; Drug or chemical Tissue Distribution; Engineering; Gene Expression; Gene Transfer; Genes; Genetic; Genetic Transcription; Grant; In Vitro; Individual; Internal Ribosome Entry Site; Ligand Binding Domain; Ligands; Mammalian Cell; Mammals; Methylation; Methyltransferase; Molecular; Mus; Nuclear Receptors; Pathway interactions; Personal Satisfaction; Phase; Plasmids; Positioning Attribute; Production; Proteins; RXR; Receptor Gene; Reporter; Reporter Genes; Research; Research Personnel; Resistance; Site; Steroids; Structure; System; Testing; Time; Transfection; Transgenic Mice; Variant; Work; design; ecdysone receptor; gene cloning; gene function; genome sequencing; histone methyltransferase; in vivo; mutant; novel; promoter; receptor; restriction enzyme; small molecule; stable cell line; therapeutic protein; tissue culture; tool; transgene expression; vector

DESCRIPTION (provided by applicant): The ability to control expression of a cloned gene both temporally and quantitatively is critical to the study of its function. For two interacting genes or gene products, the ability to control both the timing and level of expression, independently, is essential. A research tool that performed this function would be novel, timely and of significant value to the research community. Toward this end, we have identified two ecdysone receptor (EcR) Ligand Binding Domain (LBD) mutants that bind two different small molecule agonist ligands, orthogonally. Each EcR(LBD) mutant has been fused to a different DNA Binding Domain (DBD) motif, GAL4 DBD and LexA DBD. Using these EcR fusions we have demonstrated their ability to control gene expression of two reporter genes in mammalian tissue culture, independently in a ligand- dependent manner. We propose here to further test the system in a variety of mammalian cell lines under different conditions. We also propose to engineer the components into a simple, two-plasmid format, with one plasmid containing all of the components that drive the system and the other plasmid acting as a cloning vector for two genes. Positive control reporter vectors and stable cell lines will also be developed to complement it. In Phase II we will develop the in vivo use of RheoPlex by determining the tissue distribution of the two ligands, activity of the receptors and construct transgenic mice. We will also use RheoPlex to explore the coordination of DNMT1 and G9a in methylating chromatin during DNA replication. 1 Precise temporal, spatial and quantitative control over the expression of transgenes via external application of small molecules is an extremely powerful tool for gene function analysis both in vitro and in vivo. In addition, it can greatly facilitate the production of therapeutic proteins and would be crucial for gene and cell therapy applications. With the recent availability of the genome sequences of a variety of mammals, scientific attention has transferred from gene identification to functional studies. The tool under development will provide cell and molecular biologists with a unique and valuable tool to study the function of more than one gene simultaneously within the same cell. This will greatly facilitate the in vitro and in vivo study of interacting genes, gene products, genetic pathways and biochemical pathways.

描述（由申请人提供）：在时间上和定量上控制克隆基因表达的能力对于研究其功能至关重要。对于两个相互作用的基因或基因产物，独立控制表达的时间和水平的能力是必不可少的。一个能够履行这一职能的研究工具将是新颖的、及时的，对研究界具有重大价值。为此，我们已经确定了两个蜕皮激素受体（EcR）配体结合结构域（LBD）的突变体，结合两个不同的小分子激动剂配体，正交。每个EcR（LBD）突变体已经融合到不同的DNA结合结构域（DBD）基序，GAL 4 DBD和莱克萨DBD。使用这些EcR融合体，我们已经证明它们能够以配体依赖性方式独立地控制哺乳动物组织培养物中两个报告基因的基因表达。我们建议在不同条件下在各种哺乳动物细胞系中进一步测试该系统。我们还建议将组件工程化为一个简单的双质粒格式，其中一个质粒包含驱动系统的所有组件，另一个质粒作为两个基因的克隆载体。在第二阶段，我们将通过测定两种配体的组织分布、受体的活性和构建转基因小鼠来开发Rheoalbumin的体内应用。我们还将使用Rheoplastics来探索DNMT1和G9a在DNA复制过程中甲基化染色质的协调。1通过外部应用小分子对转基因表达进行精确的时间、空间和定量控制是体外和体内基因功能分析的极其强大的工具。此外，它可以极大地促进治疗性蛋白质的生产，对于基因和细胞治疗应用至关重要。随着最近各种哺乳动物基因组序列的可用性，科学的注意力已经从基因鉴定转移到功能研究。正在开发的工具将为细胞和分子生物学家提供一种独特而有价值的工具，用于研究同一细胞内多个基因的功能。这将极大地促进相互作用基因、基因产物、遗传途径和生化途径的体外和体内研究。