Stable and heritable cell-specifc knock down of gene expression in C. elegans

线虫中基因表达的稳定且可遗传的细胞特异性敲低

• 批准号: 8537513
• 负责人: DANIEL L CHASE
• 金额: $ 5.83万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-28 至 2014-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8537513
• 关键词: 3' Untranslated Regions; Adopted; Affect; Animal Model; Animals; Apoptosis; Behavioral; Behavioral Assay; Biochemical; Biological Process; C. elegans genome; Caenorhabditis elegans; Cell division; Cells; DNA biosynthesis; Defect; Development; Dissection; Effectiveness; Eukaryotic Cell; Frameshift Mutation; Gene Expression; Gene Targeting; Genes; Genetic Models; Goals; Health; Homologous Protein; Human; Individual; Inherited; Letters; Light; Measures; Mediating; Messenger RNA; Methods; Molecular; Muscle function; Mutation; Nematoda; Neurons; Nonsense Codon; Nonsense Mutation; Nonsense-Mediated Decay; Organism; Physiological; Pilot Projects; Process; Proteins; RNA; RNA Interference; RNA Processing; RNA Splicing; Refractory; Reporter; Reporter Genes; Research; Research Personnel; Signal Transduction; Study Section; Testing; Time; Transcript; Work; animal population study; cell type; design; gene function; genetic manipulation; insight; interest; knock-down; mRNA Expression; mRNA Transcript Degradation; mutant; neural circuit; neurogenesis; neurotransmission; promoter; protein function; research study; tool; transgene expression

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to develop a method for heritable, cell-specific knock down of gene expression in the nematode C. elegans. The ability to knock down gene expression in individual cell types in this organism will allow a more detailed understanding of protein function and will permit the dissection of cellular interactions such as those present in neural circuits. Our strategy has several advantages over current knock down methods and it usurps the nonsense-mediated decay (NMD) machinery that is present in all eukaryotic cells designed to degrade mRNA transcripts containing premature termination codons. In our strategy we express the gene of interest at endogenous levels with a "degradation tag" - a 3'-untranslated region (3'-UTR) that targets the mRNA transcript for degradation in wild-type cells but not in cells in which the NMD machinery has been compromised by mutation. Using cell-specific promoters we then convert NMD- deficient cells into NMD-competent cells to cause cell-specific degradation of mRNA transcripts containing the degradation tag. To provide proof that our method will work, we provide preliminary results in which we have fused a heterologous NMD degradation tag onto a reporter gene and show NMD-dependent knock down of reporter mRNA expression. We also show that we can knock down the function of an endogenous C. elegans gene using this heterologous degradation tag to cause behavioral defects similar to those observed in null mutants. In this proposal we plan to replace the heterologous degradation tag used in pilot studies with gene- specific degradation tags that will preserve the gene's 3'-UTR necessary for normal expression and stability. Thus in this proposal we plan to: 1) Develop and test gene-specific degradation tags for knock down and use them to knock down expression of endogenous genes in specific cell types while preserving wild-type expression in all other cells; 2) Quantitate transgene expression and knock down efficiency; and 3) compare the efficiency of our knock down method with the other heritable method (hairpin RNA, hpRNA) using unc-4 (a gene largely refractory to knock down by all other methods) as our target gene. Unlike other methods of gene knock down, our strategy does not rely on RNA interference and thus there is absolutely no spreading of the knock down effects to other cells. Our strategy can knock down the expression of any gene in any cell type and thus will likely be adopted by most C. elegans researchers. Because many of the proteins expressed in C. elegans are homologous to proteins expressed in humans (~40% of all proteins) we expect that the analysis of protein function in C. elegans using our cell-specific knock down strategy will shed new light on the physiological function of homologous proteins in humans.

描述（由申请人提供）：本提案的长期目标是开发一种用于线虫C中基因表达的可遗传、细胞特异性敲低的方法。优雅的在这种生物体中敲低单个细胞类型中基因表达的能力将允许更详细地了解蛋白质功能，并允许解剖细胞相互作用，例如存在于神经回路中的细胞相互作用。我们的策略与目前的敲除方法相比具有几个优点，并且它篡夺了所有真核细胞中存在的无义介导的衰变（NMD）机制，该机制旨在降解含有提前终止密码子的mRNA转录物。在我们的策略中，我们用“降解标签”在内源水平表达感兴趣的基因--一个3 '-非翻译区（3'-UTR），其靶向mRNA转录物在野生型细胞中降解，但不在NMD机制已被突变损害的细胞中降解。然后使用细胞特异性启动子，我们将NMD缺陷细胞转化为NMD感受态细胞，以引起含有降解标签的mRNA转录物的细胞特异性降解。为了证明我们的方法将起作用，我们提供了初步结果，其中我们将异源NMD降解标签融合到报告基因上，并显示NMD依赖性敲低报告mRNA表达。我们还表明，我们可以敲低功能的内源性C。elegans基因使用这种异源降解标签导致与在无效突变体中观察到的那些类似的行为缺陷。在该提议中，我们计划用基因特异性降解标签替换先导研究中使用的异源降解标签，所述基因特异性降解标签将保留正常表达和稳定性所必需的基因的3 '-UTR。因此，在本提案中，我们计划：1）开发和测试用于敲除的基因特异性降解标签，并使用它们来敲除特定细胞类型中内源基因的表达，同时保留所有其他细胞中的野生型表达; 2）定量转基因表达和敲除效率;和3）比较我们的敲除方法与使用unc-4（一种很大程度上难以被所有其他方法敲除的基因）作为我们的靶基因的其他可遗传方法（发夹RNA，hpRNA）的效率。与其他基因敲除方法不同，我们的策略不依赖于RNA干扰，因此绝对不会将敲除效应扩散到其他细胞。我们的策略可以在任何细胞类型中敲低任何基因的表达，因此很可能被大多数C。elegans研究人员。由于C.秀丽隐杆线虫与人类表达的蛋白质同源（约占所有蛋白质的40%），我们期望C.使用我们的细胞特异性敲低策略的线虫将为人类同源蛋白质的生理功能提供新的线索。