Gene Targeting in Flies and Worms with ZFNs

描述(由申请人提供)： 基因打靶是一种将现有的染色体片段替换为在实验室中被改变的相应片段的过程。这使得可以将设计好的突变引入模型生物的基因中，以研究基因功能。基因打靶的应用因其固有的低效而在许多情况下受到限制。已经开发出一种技术，可以在使用该技术的生物体和细胞类型中实现高效的基因靶向。它是基于被称为锌指核酸酶(ZFN)的工程酶直接切割所需的靶标。通过操纵锌指上的关键残基，许多不同的DNA序列可以被识别和切割，这种切割激活了与设计的供体DNA进行同源重组的靶标。在对整个果蝇和培养的哺乳动物细胞进行的实验中，基因靶向的频率达到了20%。这项建议是为了进一步探索ZFN诱导的基因打靶在果蝇中的能力和实用性，并将这一过程推广到线虫。在果蝇上的实验将测试靶向效率对同源性的数量和引入的改变类型的依赖；测试一种非常有希望的方法，以降低一些ZFN观察到的部分致死性；测试靶向比迄今已实现的更广泛的序列的能力；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，这一过程处于发育的早期阶段。ZFN诱导的合成靶点的切割已经在体细胞中实现。这项提议是为了证明先前存在的染色体位点的分裂；测试允许ZFN胚系表达的程序；以及证明在蠕虫中的胚系分裂和基因替换。在这两种流行的模式生物中发展有效的基因打靶将使基因功能的探索更加有效。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双链断裂基本上在所有生物体中都遵循类似的途径。基因打靶可以用来建立人类疾病的动物模型，并最终用于人类的基因治疗。

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.