Engineering the C. elegans Genome

描述（由申请人提供）：基因分析依赖于随意引入、消除或修饰基因的能力。这种技术在酵母、苍蝇和老鼠等遗传模式生物中是先进的，但在最常用的模式生物之一秀丽隐杆线虫中是有限的。在蠕虫中，这种方法有些粗糙：基因敲除是通过随机突变来实现的，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来实现的。尽管存在这些限制，线虫的研究在过去的35年里为生物学做出了一些最重要的发现：Ras - MAP激酶途径、细胞死亡途径、RNA干扰和microRNAs的转录后调控都是例子。我们建议开发在秀丽隐杆线虫基因组中插入、删除或修饰基因的技术。这些技术依靠调动转座子来改造基因组；这些方法将成为整个秀丽隐杆线虫研究界的资源。目的1。改进的单副本插入。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。通用插入点。我们将在所有染色体上生成与单个靶向质粒兼容的通用插入位点。这将大大增加该技术的多功能性。目标3。基因打靶。我们将开发一种策略，在其内源性环境中操纵基因。这项技术将允许研究人员设计突变，包括基因敲除，除了预期的突变外，基因中没有任何外来的DNA变化。

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.