Engineering the C. elegans Genome

描述（由申请人提供）：遗传分析依赖于随意引入、消除或修饰基因的能力。这种技术在遗传模式生物如酵母、苍蝇和小鼠中是先进的，但在蠕虫C中是有限的。线虫，这是最常用的模式生物之一。在蠕虫中，这种方法有些粗糙：基因敲除是通过随机诱变，恢复基因功能是通过多拷贝染色体外阵列或随机基因整合。尽管有这些限制C。在过去的35年里，对线虫的研究促成了一些生物学上最重要的发现：Ras - MAP激酶途径、细胞死亡途径、RNA干扰和microRNA的转录后调节都是例子。 我们建议开发技术，插入，删除或修改基因在C。线虫基因组这些技术依赖于调动转座子来设计基因组;这些方法将成为整个C。elegans研究社区目标1.改进的单拷贝插入。我们将描述每条染色体上的插入位点，并提高转基因插入的效率。我们还将设计可用于野生型背景的瞬时选择试剂。目标2.通用插入位点。我们将在所有染色体上产生通用插入位点，这些位点将与单个靶向质粒兼容。这将大大增加该技术的通用性。目标3。基因靶向。我们将开发一种策略来操纵基因在其内源性的背景。这项技术将允许研究人员设计突变，包括敲除，除了预期的突变外，基因中没有任何外来的DNA变化。 公共卫生相关性：C.线虫是细胞生物学研究的主要模式生物之一，300多种C.美国的线虫实验室目前接受美国国立卫生研究院的资助。这些实验室从事研究衰老、癌症、毒理学、神经科学和对病原体反应的基因的项目。修改C.无限制的线虫基因组将使实验更快，结果更可靠，用于人类健康各个方面的研究。

DESCRIPTION (provided by applicant): Genetic analysis relies on the ability to introduce, eliminate or modify genes at will. Such techniques are advanced in genetic model organisms such as yeast, flies and mice, but are limited in the worm C. elegans, which is one of the most commonly used model organisms. In worms such methods are somewhat crude: gene knockouts are by random mutagenesis and restoring gene function is via multicopy extrachromosomal arrays or random gene integrations. Despite these limitations C. elegans research has contributed to some of the most important discoveries in biology in the last 35 years: Ras - MAP kinase pathways, cell death pathways, RNA interference and posttranscriptional regulation by microRNAs are examples. We propose to develop techniques to insert, delete or modify genes in the C. elegans genome. The techniques rely on mobilizing transposons to engineer the genome; the methods will be a resource for the whole C. elegans research community. Aim 1. Improved single copy insertion. We will characterize insertion sites on each chromosome and increase the efficiency of transgene insertions. We will also devise transient selection reagents that can be used in a wild-type background. Aim 2. Universal insertion sites. We will generate universal insertion sites on all chromosomes that will be compatible with a single targeting plasmid. This will substantially increase the versatility of the technique. Aim 3. Gene targeting. We will develop a strategy to manipulate genes in their endogenous context. This technique will allow researchers to engineer mutations, including knock-outs, without any extraneous DNA changes in the gene except the intended mutation. PUBLIC HEALTH RELEVANCE: C. elegans is one of the major model organisms for studies of cell biology; more than 300 C. elegans labs in the US currently receive funding from the NIH. These labs pursue projects studying genes involved in aging, cancer, toxicology, neuroscience, and response to pathogens. The ability to modify the C. elegans genome without limits will make experimentation much faster and results more reliable for studies of all aspects of human health.