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

项目摘要 该提案的长期目标是开发一种稳定的、可遗传的和细胞特异性敲除的方法， 基因在线虫C.优雅敲低单个细胞中基因表达的能力 这种生物体中的蛋白质类型将允许更详细地了解单个细胞中的蛋白质功能， 允许解剖细胞的相互作用，如那些存在于神经回路。我们的战略篡夺了 无义介导的衰变（NMD）机制存在于所有真核细胞中，旨在降解mRNA 含有提前终止密码子的转录物。在我们的策略中，我们用一个3 '- 非翻译区（UTR），其靶向mRNA转录物以在野生型细胞中降解，但不在细胞中降解。 国家导弹防御系统的机器已经被突变破坏了。利用细胞特异性启动子， 将NMD缺陷细胞转化为NMD感受态细胞，以引起mRNA转录物的细胞特异性降解 含有针对3 '-UTR的NMD。我们将证明这种方法的可行性，1）敲下 表达编码荧光蛋白的多拷贝转基因，和2）敲低编码荧光蛋白的多拷贝转基因的表达， 内源信号基因我们将定量这些敲低细胞中mRNA和蛋白质的量。这 该策略可用于敲低生物体中任何细胞中任何基因的表达。因为许多 C.线虫有哺乳动物的直系同源物（约40%的基因），我们的敲除策略 这将有助于更好地了解人类蛋白质的功能，这将对人类的健康产生深远的影响。 人体健康举个例子，了解神经递质受体及其细胞内信号传导 伴侣在特定细胞类型中的功能可以导致更好的治疗疾病状态，如帕金森氏症 精神分裂症和药物滥用。

Project Summary The long-term goal of this proposal is to develop a method for stable, heritable and 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 in individual cells and will permit the dissection of cellular interactions such as those present in neural circuits. Our strategy usurps the nonsense-mediated decay (NMD) machinery present in all eukaryotic cells designed to degrade mRNA transcripts that contain premature termination codons. In our strategy we express the gene of interest with a 3'- untranslated region (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 can then convert NMD deficient cells into NMD competent cells to cause cell-specific degradation of mRNA transcripts that contain NMD targeting 3'-UTRs. We will demonstrate the feasibility of this method by 1) knocking down the expression of a multicopy transgene encoding a fluorescent protein and 2) knocking down the expression of an endogenous signaling gene. We will quantify the amount of mRNA and protein in these knock down cells. This strategy can be used to knock down the expression of any gene in any cell in the organism. Because many of the proteins expressed in C. elegans have mammalian orthologs (~40% of all genes) our knock down strategy should lead to a better understanding of protein function in humans that will have far-reaching implications on human health. As one example, understanding how neurotransmitter receptors and their intracellular signaling partners function in specific cell types can lead to better therapies for disease states such as Parkinson's disease, schizophrenia and drug abuse.