Zinc Finger Targeting of C. elegans Genes

描述(由申请人提供)：线虫是一种强大和成熟的模式生物，用于研究发育、细胞凋亡、细胞增殖、衰老和神经疾病潜在的基本和保守的分子机制。然而，在线虫中，通过其他生物中使用的基因打靶方法进行内源染色体基因的定向突变或定点插入转基因是非常低效的，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研究表明，在感兴趣的序列上引入双链断裂(DSB)可以刺激许多数量级的基因靶向性。在这些研究中，使用锌指核酸酶(ZFN)将DSB引入特定的基因组序列，ZFN是一种人造限制性内切酶，可以被改造成切割特定的靶DNA序列。这个探索性的R21建议开发ZFN刺激的基因靶向用于线虫。该项目的长期目标包括建立线虫基因打靶和基因失活的最佳技术，以及为更广泛的学术研究社区提供设计的锌指蛋白用于基因打靶的策略。这项建议的具体目的是：(1)测试ZFN是否可用于将DSB导入整合的GFP报告基因；以及(2)测试ZFN增强的基因打靶是否可用于创建特定突变或将转基因(如GFP)引入线虫种质中的特定内源基因。所提出的研究将为线虫的研究提供一种强大而新颖的基因技术。加速和扩大线虫的研究将直接提高在这一成熟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线虫研究人员能够通过在线虫的人类疾病相关蛋白的同源物中插入特定的变化来建立更准确的人类疾病模型。我们预计，基于ZFN的基因打靶技术的发展将导致对人类疾病潜在基本机制的更快理解。公共卫生相关性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。对线虫遗传和发育过程的研究已经提供了重要的见解，然而，缺乏一种易操作的方法来操纵内源基因结构一直是一个限制。用于操纵线虫内源序列的锌指核酸酶技术的发展，将通过允许对人类遗传病进行更精确的建模，大大增强这一本已强大的系统。

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.