Targeted genome modification of guinea pig and sheep using engineered zinc finger

描述（由申请方提供）：拟定研究的目的是开发新方法和/或调整当前可用的技术，用于对各种实验室动物种属进行遗传修饰。目前，小鼠是研究中最常见的动物物种。它们的受欢迎程度源于它们的小尺寸，繁殖和维持的相对成本低，妊娠期短，窝仔数大，以及大量的商业可用试剂和技术。动物模型开发的最大进步之一是产生表达外源DNA的小鼠（转基因小鼠，敲入小鼠）或具有天然基因缺失的小鼠（敲除或基因缺陷小鼠）的技术。使用小鼠作为动物模型的一个主要缺点是，它们通常不能可靠地模拟人类发育或疾病。然而，许多用于修饰小鼠基因组的技术难以适应非啮齿动物物种，或者在其他物种中尝试成本过高。已经开发了几种方法来克服这些障碍，并且最近的几篇报道描述了一种使用锌指核酸酶（ZFN）产生基因缺陷大鼠的非常成功的方法。简而言之，ZFN是由融合至内切核酸酶的工程化DNA结合结构域组成的合成蛋白，其诱导特异性DNA序列中的双链断裂。DNA断裂通过宿主细胞修复途径修复，其中一部分修复包含导致基因突变的错误，这些基因突变通常实际上转化为靶基因产物的缺失。该项目的总体目标是使用ZFN技术开发可靠且具有成本效益的方法，用于生产适用于不同物种的转基因动物。精子介导的基因转移（SMGT）将用于将ZFN递送到发育中的胚胎。使用该方法，将新鲜分离的精子与编码ZFN的DNA一起孵育。DNA与精子表面的特定蛋白质结合，并在细胞内转运。载有DNA的精子可以在体外或体内使卵子受精。这项技术将在两个物种（一个啮齿动物和一个非啮齿动物）中进行比较，以证明概念的证明，并产生转基因豚鼠和绵羊，这些豚鼠和绵羊是人类疾病的宝贵模型。该项目的具体目标是：（1）产生免疫反应的一个或多个基因缺陷的豚鼠;（2）产生心脏和呼吸系统胚胎发育的一个或多个基因缺陷的绵羊。

DESCRIPTION (provided by applicant): The objective of the proposed research is to develop novel approaches and/or adapt currently available techniques for genetic modification of a variety of laboratory animal species. Currently, mice are the most common species of animal used in research. Their popularity derives from their small size, low relative cost to breed and maintain, short gestation, and large litter size, as well as the vast array of commercially-available reagents and technologies. One of the greatest advances in animal model development has been the generation of techniques for producing mice that express foreign DNA (transgenic mice, knock-in mice) or have a native gene deleted (knock-out, or gene-deficient, mice). A major drawback to using mice as animal models is that they often fail to reliably mimic human development or disease. However, many of the techniques used to modify the mouse genome have been difficult to adapt to non-rodent species or are cost prohibitive to attempt in other species. Several methods have been developed to overcome these obstacles, and several recent reports have described a highly successful method for generating gene deficient rats using zinc finger nucleases (ZFNs). Briefly, ZFNs are synthetic proteins consisting of an engineered DNA- binding domain fused to an endonuclease that induce double-stranded breaks in specific DNA sequences. The DNA breaks are repaired by host cell repair pathways, with a proportion of the repairs incorporating errors resulting in genetic mutations that often translate, in effect, to deletion of the targeted gene product. The overall goal of the project described here is to use ZFN technology in the development of reliable and cost-effective methods for producing genetically modified animals that is applicable across species. Sperm- mediated gene transfer (SMGT) will be used to deliver the ZFN to the developing embryo. Using this method, freshly isolated sperm will be incubated with DNA encoding the ZFN. DNA binds to specific proteins on the surface of the sperm and is transported intracellularly. DNA-laden sperm can then be used to fertilize ova in vitro or in vivo. This technology will be compared in two species, one rodent and one non-rodent, to demonstrate proof of concept and to generate genetically modified guinea pigs and sheep that are valuable models for human diseases. The specific aims of the project are to generate 1) guinea pigs deficient in one or more genes involved in the generation of immune responses and 2) sheep deficient in one or more genes involved in embryonic development of the cardiac and respiratory systems.