Stimulating Gene Targeting with Zinc Finger Nucleases

描述（由申请人提供）：虽然靶向基因置换是 可能在一些生物体中，包括小鼠细胞，这种低效率 限制了一些应用。此外，没有有效的基因靶向 对于许多实验生物体存在协议。造成双链断裂 刺激基因定位。说这一点是为 测试具有锌指DNA结合结构域的核酸酶的能力 （ZFN）作为靶向DNA切割试剂。锌指能识别新的 序列可以通过改变一些氨基酸的同一性来产生， 直接接触DNA，因此原则上它们能够被引导到 任意选择目标。基因打靶技术的研究进展 切割靶应该适用于许多不同的生物体， 情况，包括人类疾病的动物模型的生成， 最终应用于人类基因治疗。 ZFN在哺乳动物细胞中的基因靶向应用将继续进行 最初定位于小鼠HPRT基因，这对于遗传分析具有优势。 识别外显子3中的靶标的锌指已经被分离， 工程化到核酸酶构建体中。用于递送这些的各种方法 将测试ZFN和培养ES细胞的标记的供体DNA， 测量靶向重组的效率。的程度的影响 将评估供体和靶DNA之间的同源性，以及位置 供体中切割位点的改变。以确定 什么样的重组途径负责ZFN切割的靶向， 通过同源重组影响DNA修复的突变的影响 会得到考验为了减少捐助者的参与， 非同源位点，参与非同源修复的基因突变将 追究为了证明这种方法的通用性， 靶向，它将被应用到另一个小鼠基因座，Rpo 2 -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.