Gene Targeting in Flies and Worms with ZFNs

描述（由申请人提供）： 基因靶向是一个程序，其中现有的染色体片段被替换为在实验室中已经改变的相应片段。这允许将设计的突变引入模式生物的基因中，以研究基因功能。基因打靶的应用在许多情况下受到其固有的低效率的限制。已经开发了一种技术，该技术允许在使用该技术的生物体和细胞类型中进行高效基因靶向。它基于用称为锌指核酸酶（ZFN）的工程化酶对所需靶标的定向切割。通过操纵锌指中的关键残基，可以识别和切割许多不同的DNA序列，并且这种切割激活靶与设计的供体DNA进行同源重组。在用整个果蝇和培养的哺乳动物细胞进行的实验中，基因靶向的频率已达到20%。本研究旨在进一步探索ZFN诱导的果蝇基因打靶的能力，并将其应用于线虫线虫。果蝇实验将测试靶向效率对同源性和引入的改变类型的依赖性;测试一种非常有前途的方法，用于减少用一些ZFN观察到的部分致死性;测试靶向比迄今为止所实现的更广泛序列的能力;并尝试简化实验程序，使其易于为所有研究人员所用。在线虫中，该过程处于发育的早期阶段。ZFN诱导的合成靶的切割已经在体细胞中实现。该提案旨在证明预先存在的染色体位点的切割;测试允许ZFN生殖系表达的程序;并证明蠕虫中的生殖系切割和基因置换。在这两种流行的模式生物中开发有效的基因靶向技术将使基因功能的探索更加有效。所吸取的经验教训将适用于其他系统，因为通过同源重组修复DNA双链断裂遵循基本上所有生物体中的类似途径。基因靶向可用于创建人类疾病的动物模型，并最终用于人类基因治疗。

DESCRIPTION (provided by applicant): Gene targeting is a procedure in which an existing chromosomal segment is replaced with a corresponding segment that has been altered in the laboratory. This allows introduction of designed mutations into genes of model organisms for investigation of gene function. Application of gene targeting has been limited in many instances by its inherent inefficiency. A technique has been developed that allows high efficiency gene targeting in the organisms and cell types where it has been used. It is based on directed cleavage of the desired target with engineered enzymes called zinc finger nucleases (ZFNs). By manipulation of key residues in the zinc fingers, many different DNA sequences can be recognized and cleaved, and this cleavage activates the target for homologous recombination with the designed donor DNA. In experiments with whole fruit flies and cultured mammalian cells, frequencies of gene targeting up to 20% have been achieved. This proposal is to further explore the capabilities and enhance the utility of ZFN-induced gene targeting in flies and to extend the procedure to the nematode, Caenorhabditis elegans. Experiments in flies will test the dependence of targeting efficiency on the amount of homology and the types of alterations introduced; test a very promising approach for reducing the partial lethality that has been observed with some ZFNs; test the ability to target a wider range of sequences than has been achieved to date; and attempt to simplify the experimental procedure to make it readily accessible to all researchers. In nematodes, the procedure is at a much earlier stage of development. ZFN-induced cleavage of synthetic targets has been achieved in somatic cells. The proposal is to demonstrate cleavage of a pre-existing chromosomal locus; to test procedures that will allow germline expression of the ZFN; and to demonstrate germline cleavage and gene replacement in the worms. Development of effective gene targeting in these two popular model organisms will make the exploration of gene function more efficient. The lessons learned will be applicable to other systems, since the repair of DNA double-strand breaks by homologous recombination follows similar pathways in essentially all organisms. Gene targeting can be used to create animal models for human disease and, ultimately, for human gene therapy.