Gene Targeting Using Modular Zinc Finger Nulceases

描述（由申请人提供）：研究生物过程的一种基本方法是通过功能丧失研究。这些努力的目标是通过破坏基因来确定基因的活性，并监测其对生物系统的影响。近年来，利用锌指核酸酶（zinc finger nucleases, ZFNs）进行基因靶向修饰的方法得到了发展。该方法涉及工程锌指DNA结合域来识别特定的目标序列。然后将这些结构域连接到FOK1核酸外切酶结构域以产生融合蛋白，该融合蛋白可以在锌指结合位点附近诱导双链DNA断裂。ZFNs已经成功地用于从植物到哺乳动物的各种系统中破坏基因。到目前为止，只有相对较少的实验室报告成功地使用了zfn。由于难以生成与特定靶序列结合的锌指结构域，以及商用ZFNs的高成本，ZFNs的使用受到限制。在初步研究中，计算机辅助“模块化”设计已成为设计功能性zfn的有效手段。本应用程序中的实验评估了使用模块化ZFN设计以斑马鱼为模型系统产生种系突变的策略的有效性。斑马鱼是一个强大的遗传、细胞和分子研究系统，常规进行基因靶向研究的能力将是一个重大进步。本应用中的方法广泛适用于细胞培养系统和模式生物的生物医学研究。

DESCRIPTION (Provided by Applicant): One fundamental method to investigate biological processes is by loss-of-function studies. The goal of these efforts is to determine the activity of a gene by disrupting that gene and monitoring the effects on a biological system. Recently, gene targeting technology has been developed using zinc finger nucleases (ZFNs) as a method to mutate specific genes. The approach involves engineering zinc finger DNA binding domains to recognize specific target sequences. These domains can then be tethered to the FOK1 exonuclease domains to generate a fusion protein that can induce double stranded DNA breaks near the zinc finger binding sites. ZFNs have been successfully used to disrupted genes in a variety of systems ranging from plants to mammals. Thus far, only a relatively small number of laboratories have reported successful efforts to employ ZFNs. The use of ZFNs has been curtailed by the difficulty in generating zinc finger domains that bind to specific target sequences, and by the high cost of commercially available ZFNs. In preliminary studies, computer aided "modular" design has been an effective means to engineer functional ZFNs. The experiments in this application evaluate the effectiveness of a strategy to use modular ZFN design to generate germ-line mutations using zebrafish as a model system. Zebrafish is a powerful system for genetic, cellular, and molecular studies, and the ability to routinely perform gene targeting studies would be a major advance. The methods in this application are broadly applicable to biomedical research in both cell culture systems and model organisms. RELEVANCE: The human genome project has identified thousands of novel genes, but the activity of many of these genes remains unknown. The ability to disrupt specific genes is a powerful means to determine the function of these genes. The experiments in this application evaluate the effectiveness of one means to accomplish this goal using model organisms and cell culture systems.