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

描述（由申请人提供）：本提案的长期目标是开发一种可遗传的，细胞特异性敲低线虫基因表达的方法。在这种生物体中，敲低单个细胞类型的基因表达的能力将使人们对蛋白质功能有更详细的了解，并将允许解剖细胞相互作用，如神经回路中存在的相互作用。我们的策略与目前的敲除方法相比有几个优势，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过早终止密码子的mRNA转录本。在我们的策略中，我们在内源性水平上用“降解标签”表达感兴趣的基因——一个3'-未翻译区（3'- utr），在野生型细胞中靶向mRNA转录物降解，而在NMD机制因突变而受损的细胞中不靶向。利用细胞特异性启动子，我们将NMD缺陷细胞转化为NMD胜任细胞，导致含有降解标签的mRNA转录物的细胞特异性降解。为了证明我们的方法是有效的，我们提供了初步的结果，我们将一个异源的NMD降解标签融合到一个报告基因上，并显示了NMD依赖性的报告mRNA表达的下调。我们还表明，我们可以使用这种异源降解标签敲除内源性秀丽隐杆线虫基因的功能，从而导致类似于在零突变体中观察到的行为缺陷。在本提案中，我们计划用基因特异性降解标签取代先导研究中使用的异源降解标签，这将保留基因正常表达和稳定性所需的3'- utr。因此，在本提案中，我们计划：1)开发和测试基因特异性降解标签，并使用它们在特定细胞类型中敲除内源性基因的表达，同时在所有其他细胞中保留野生型表达；2)定量转基因表达及敲除效率；3)比较我们的敲除方法与其他可遗传方法（发夹RNA， hpRNA）的效率，使用unc-4（一种基本上难以被所有其他方法敲除的基因）作为我们的靶基因。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，因此可能会被大多数秀丽隐杆线虫研究人员采用。由于秀丽隐杆线虫中表达的许多蛋白质与人类表达的蛋白质同源（约占所有蛋白质的40%），我们期望利用细胞特异性敲低策略分析秀丽隐杆线虫中的蛋白质功能，将为人类同源蛋白的生理功能提供新的线索。

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.