Zinc Finger Targeting of C. elegans Genes

描述（申请人提供）：秀丽隐杆线虫是一种强大且成熟的模式生物，用于研究发育，凋亡，细胞增殖，衰老和神经系统疾病的基本和保守分子机制。然而，在其他生物中使用的基因靶向方法对内源性染色体基因进行靶向诱变或对转基因进行位点特异性插入，在秀丽隐杆线虫中效率非常低，因此大多数研究人员无法实现。然而，最近对果蝇、植物和哺乳动物细胞的研究表明，在感兴趣的序列上引入双链断裂（DSB）可以提高许多数量级的基因靶向率。在这些研究中，利用锌指核酸酶（ZFNs）将dsb引入特定的基因组序列，锌指核酸酶是一种人工限制性内切酶，可以切割特定的目标DNA序列。这个探索性的R21提出开发zfn刺激的基因靶向用于秀丽隐杆线虫。该项目的长期目标包括建立线虫基因靶向和基因失活的最佳技术，以及为更广泛的学术研究界提供设计锌指蛋白的基因靶向策略。本文的具体目的是：(1)测试ZFNs是否可以用于将DSB引入整合的GFP报告基因中；(2)测试zfn增强基因靶向是否可用于产生特定突变或将转基因（如GFP）引入秀丽隐杆线虫种系中的特定内源基因。所提出的研究将为秀丽隐杆线虫的研究提供一种强大而新颖的遗传技术。加速和扩大秀丽隐杆线虫的研究将直接提高分析的速度和能力，可以在这种成熟的模式生物中进行。高效基因靶向技术的发展将允许秀丽隐杆线虫研究人员通过在秀丽隐杆线虫的人类疾病相关蛋白同源物中插入特定的变化，建立更准确的人类疾病模型。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的基本机制的更快速的理解。在其他脊椎动物或无脊椎动物系统中对人类疾病进行建模需要精确改变基因结构的技术。秀丽隐杆线虫的遗传和发育过程的研究提供了重要的见解，然而，缺乏一种易于处理的方法来操纵内源性基因结构一直是一个限制。利用锌指核酸酶技术操纵秀丽隐杆线虫内源性序列的发展，将通过对人类遗传疾病进行更精确的建模，大大增强这一已经强大的系统。

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.