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

项目概述：拟议研究的目标是开发新的方法和/或适应 目前可用于多种实验室动物物种的遗传修饰的技术。目前， 老鼠是研究中最常见的动物种类。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，产仔数大，以及大量的 市售试剂和技术。动物模型最大的进步之一 已经发展了产生表达外源DNA（转基因）的小鼠的技术 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）。的主要缺点 使用小鼠作为动物模型的一个缺点是，它们往往不能可靠地模拟人类的发育或疾病。然而，在这方面， 许多用于修改小鼠基因组的技术很难适用于非啮齿类动物 或者在其它物种中尝试成本过高。已经开发了几种方法来克服这些问题 障碍，并且最近的几份报告描述了一种非常成功的方法，用于产生基因缺陷的 大鼠使用锌指核酸酶（ZFN）。简而言之，ZFN是由工程化DNA组成的合成蛋白质。 与内切核酸酶融合的结合结构域，其诱导特定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错误 导致基因突变，其通常实际上翻译为靶基因产物的缺失。 本文所述项目的总体目标是使用ZFN技术开发可靠且 生产适用于所有物种的转基因动物的具有成本效益的方法。精子- 介导的基因转移（SMGT）将用于将ZFN递送至发育中的胚胎。使用这种方法， 将新鲜分离的精子与编码ZFN的DNA一起孵育。DNA与细胞表面的特定蛋白质结合， 精子表面，并在细胞内运输。然后，携带DNA的精子可以用来使卵子受精， 体外或体内。这项技术将在两个物种中进行比较，一个啮齿动物和一个非啮齿动物， 展示概念证明，并产生有价值的转基因豚鼠和绵羊 人类疾病的模型。该项目的具体目标是：（1）培养缺乏一种或 更多的基因参与产生免疫反应和2）绵羊缺乏一个或多个基因 参与心脏和呼吸系统的胚胎发育。

Project summary: 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.