Stimulating Gene Targeting with Zinc Finger Nucleases

描述(申请人提供)：虽然有针对性的基因替换是 在包括小鼠细胞在内的一些生物体中可能存在这种低效率 进程限制了一些应用程序。此外，没有有效的基因靶向 协议适用于许多实验生物体。制造双链断裂 在染色体中，DNA刺激基因靶向。这项建议的目的是 检测含锌指DNA结合区的核酸酶的功能 (ZFns)作为靶向DNA切割试剂。识别新产品的锌手指 序列可以通过改变几个氨基酸的同一性来产生 直接与DNA联系，所以原则上他们能够被定向到 任意选择的目标。刺激基因打靶的途径 切割目标应该适用于许多不同的生物体和 情况，包括人类疾病动物模型的产生和 最终发展到人类基因治疗。 ZFN在哺乳动物细胞基因打靶中的应用将被继续进行 最初是在小鼠的HPRT基因上，这对遗传分析具有优势。 识别外显子3中的靶的锌指已经被分离出来 被改造成核酸酶结构。实现这些目标的各种方法 将对ZFN和标记的ES细胞供体DNA进行测试，并 测定靶向重组的效率。影响程度的程度 供体和目标DNA之间的同源性将被评估，位置也将被评估 供体相对于卵裂部位的改变。要确定 哪些重组途径负责ZFN的靶向切割， 通过同源重组影响DNA修复的突变效应 将会受到考验。为了减少捐赠者在 非同源位点，参与非同源修复的基因突变将 被调查。为了证明这种方法对基因的普遍性 靶向，它将被应用于另一个小鼠基因，Rpo2-1基因。

DESCRIPTION (provided by applicant): Although targeted gene replacement is possible in some organisms, including mouse cells, the low efficiency of this process limits some applications. In addition, no effective gene targeting protocol exists for many experimental organisms. Making a double-strand break in chromosomal DNA stimulates gene targeting. The purpose of this proposal is to test the capabilities of nucleases with zinc finger DNA-binding domains (ZFNs) as targetable DNA cleavage reagents. Zinc fingers that recognize new sequences can be generated by changing the identity of a few amino acids that contact the DNA directly, so they are capable in principle of being directed to arbitrarily selected targets. The approach of stimulating gene targeting by cleaving the target should be applicable to many different organisms and situations, including the generation of animal models of human disease and ultimately to human gene therapy. The application of ZFNs to gene targeting in mammalian cells will be pursued initially at the mouse HPRT gene, which has advantages for genetic analysis. Zinc fingers that recognize a target in exon 3 have been isolated and engineered into the nuclease construct. Various methods for delivering these ZFNs and a marked donor DNA to cultured ES cells will be tested, and the efficiency of targeted recombination measured. The influence of the extent of homology between donor and target DNAs will be assessed, as will the location of the alteration in the donor with respect to the cleavage site. To determine what pathway of recombination is responsible for targeting with ZFN cleavage, the effects of mutations that affect DNA repair by homologous recombination will be tested. In an attempt to reduce integration of the donor at nonhomologous sites, mutations in genes involved in nonhomologous repair will be investigated. To demonstrate the generality of this approach to gene targeting, it will be applied to another mouse locus, the Rpo2-1 gene.