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: 8445729
• 负责人: DANIEL L CHASE
• 金额: $ 7.47万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8445729
• 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. PUBLIC HEALTH RELEVANCE: C. elegans has proven to be a valuable genetic model organism for study of important biological processes including neurogenesis and degeneration, developmental programming and cell death. Many of the molecular mechanisms identified in the worm that control and/or mediate these processes are conserved in humans and thus studies in C. elegans have contributed to our understanding of many human pathological conditions. The ability to stably and heritably knock down gene function in individual cells in C. elegans will allw unprecedented insight into the cell-specific function of proteins. The knock down approach described in this proposal can be used to study the function of any protein in any cell type to understand any biological process including those that directly benefit human health such as neurotransmission, muscle function, and cell division.

描述(申请人提供)：这项提案的长期目标是开发一种可遗传的、细胞特异性的敲除线虫基因表达的方法。在这种生物体中，能够降低单个细胞类型的基因表达，将使我们能够更详细地了解蛋白质的功能，并有助于剖析细胞之间的相互作用，例如那些存在于神经回路中的相互作用。我们的策略比目前的敲除方法有几个优点，它取代了所有真核细胞中存在的无义介导的衰退(NMD)机制，该机制旨在降解包含提前终止密码子的mRNA转录本。在我们的策略中，我们通过“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)，它针对野生型细胞中的mRNA转录本进行降解，而不是在NMD机制因突变而受损的细胞中。然后，使用细胞特异性启动子，我们将NMD缺陷细胞转化为NMD活性细胞，从而导致含有降解标签的mRNA转录本的细胞特异性降解。为了证明我们的方法是有效的，我们提供了初步的结果，其中我们已经将一个异源的NMD降解标签融合到一个报告基因上，并显示了NMD依赖的报告mRNA表达的下调。我们还表明，我们可以使用这个异源降解标签来敲除内源线虫基因的功能，从而导致类似于零突变体中观察到的行为缺陷。在这项建议中，我们计划用基因特定的降解标签取代在试点研究中使用的异源降解标签，以保存基因正常表达和稳定所必需的3‘-UTR。因此，在这项建议中，我们计划：1)开发和测试针对敲除的基因特异性降解标签，并使用它们来下调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)定量转基因表达和击倒效率；以及3)以UNC-4(一种在很大程度上难以被所有其他方法击倒的基因)为目标基因，将我们的击倒方法与其他可遗传方法(发夹RNA，hpRNA)的效率进行比较。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。我们的策略可以降低任何细胞类型中任何基因的表达，因此很可能会被大多数线虫研究人员采用。由于线虫中表达的许多蛋白质与人类表达的蛋白质(约占所有蛋白质的40%)是同源的，我们希望利用我们的细胞特异性敲除策略对线虫蛋白质功能的分析将为人类同源蛋白质的生理功能提供新的线索。 公共卫生相关性：秀丽线虫已被证明是一种有价值的遗传模式生物，用于研究重要的生物过程，包括神经发生和退化、发育编程和细胞死亡。在线虫中发现的许多控制和/或调节这些过程的分子机制在人类中是保守的，因此对线虫的研究有助于我们了解许多人类的病理状况。稳定和遗传地敲除线虫单个细胞中的基因功能的能力将为蛋白质的细胞特有功能提供前所未有的洞察力。这项建议中描述的击倒方法可以用于研究任何细胞类型中任何蛋白质的功能，以了解任何生物过程，包括那些直接有益于人类健康的过程，如神经传递、肌肉功能和细胞分裂。