Zinc Finger Targeting of C. elegans Genes

描述（由申请人提供）：C。秀丽线虫是一种强大的和完善的模式生物，用于研究发育、凋亡、细胞增殖、衰老和神经疾病的基本和保守的分子机制。然而，通过其他生物中使用的基因打靶方法对内源染色体基因进行定向诱变或转基因的位点特异性插入在C.因此，大多数研究人员无法获得。然而，最近在果蝇、植物和哺乳动物细胞中的工作表明，在感兴趣的序列处引入双链断裂（DSB）可以刺激基因靶向的速率许多数量级。在这些研究中，使用锌指核酸酶（ZFN）在特定基因组序列处引入DSB，所述锌指核酸酶是可以被工程化以切割特定靶DNA序列的人工限制性内切核酸酶。这个探索性的R21提出开发ZFN刺激的基因靶向用于C。优雅本项目的长期目标包括建立最佳的基因打靶和基因失活技术。elegans和建立战略，提供设计师锌指蛋白基因靶向更广泛的学术研究界。该提议的具体目的是：（1）测试ZFN是否可用于将DSB引入整合的GFP报告基因中;以及（2）测试ZFN增强的基因靶向是否可用于产生特定突变或将转基因（例如GFP）引入C.秀丽隐杆线虫生殖系本研究为C.基于Elegans的研究加速和扩大C。elegans的研究将直接提高在这种已建立的模式生物中进行分析的速度和能力。高效基因打靶技术的发展将使C。elegans研究人员通过将特定的变化插入C.人类疾病相关蛋白质的同源物。我们期望基于ZFN的基因靶向技术的发展将导致对人类疾病基本机制的更快理解。在其他脊椎动物或无脊椎动物系统中模拟人类疾病需要精确改变基因结构的技术。对C.线虫提供了重要的见解，然而，缺乏操纵内源基因结构的易处理的方法一直是一个限制。开发锌指核酸酶技术操纵C.秀丽隐杆线虫将通过允许更精确地模拟人类遗传疾病来显著增强这个已经强大的系统。

DESCRIPTION (provided by applicant): C. elegans is a powerful and well-established model organism used to investigate basic and conserved molecular mechanisms underlying development, apoptosis, cellular proliferation, aging, and neurological disease. However, targeted mutagenesis of endogenous chromosomal genes or site-specific insertion of transgenes by gene targeting methods used in other organisms is highly inefficient in C. elegans and, therefore, inaccessible to most researchers. However, recent work in Drosophila, plants, and mammalian cells has shown that introduction of a double-stranded break (DSB) at a sequence of interest can stimulate rates of gene targeting by many orders of magnitude. In these studies, DSBs were introduced at specific genomic sequences using zinc finger nucleases (ZFNs), artificial restriction endonucleases that can be engineered to cleave specific target DNA sequences. This exploratory R21 proposes to develop ZFN-stimulated gene targeting for use in C. elegans. The long-terms goals of this project include establishing optimal techniques for gene targeting and gene inactivation in C. elegans and establishing strategies to provide designer Zn-finger proteins for gene targeting for the broader academic research community. The specific aims of this proposal are: (1) to test whether ZFNs can be used to introduce a DSB into an integrated GFP reporter gene; and (2) to test whether ZFN-enhanced gene targeting can be used to create specific mutations or to introduce transgenes (e.g. GFP) into specific endogenous genes in the C. elegans germline. The proposed studies will provide a powerful and novel genetic technique for C. elegans-based research. Accelerating and expanding C. elegans research will directly increase the speed and power of the analyses that can be carried in this well-established model organism. Development of efficient gene targeting techniques will permit C. elegans researchers to establish more accurate models of human disease by inserting specific changes into C. elegans homologs of human disease-related proteins. We expect that the development of ZFN-based gene targeting techniques will result in a more rapid understanding of basic mechanisms underlying human disease. Public Health Relevance Modeling human diseases in other vertebrate or invertebrate systems requires technologies to precisely alter gene structure. Investigations of genetic and developmental processes in C. elegans have provided important insights, however, the lack of a tractable method for manipulating endogenous gene structure has been a limitation. Development of the Zinc-Finger Nuclease technology for manipulating endogenous sequences in C. elegans will significantly augment this already powerful system by allowing more precise modeling of human genetic diseases.