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

使用工程锌指对豚鼠和绵羊进行靶向基因组修饰

基本信息

批准号：
8332316
负责人：
Hilda Holcombe
金额：
$ 2.23万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-15 至 2012-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8332316
关键词：
Animal Model Animal Welfare Animals Antigen Presentation Artificial Insemination B-Cell Development Base Sequence Breeding Cardiac Cardiopulmonary Cavia Cells Chloride Ion Chlorides Cystic Fibrosis Transmembrane Conductance Regulator Cytotoxic T-Lymphocytes DNA DNA Binding DNA Binding Domain DNA Sequence Development Embryo Embryonic Development Engineering Equipment Fertilization Fertilization in Vitro Flow Cytometry Gene Mutation Gene Transfer General Hospitals Generations Genes Genetic Techniques Genetically Modified Animals Genome Goals Harvest Human Human Development Human Resources Immune response In Vitro Incubated Injection of therapeutic agent Institution Knock-in Mouse Knock-out Laboratories Laboratory Animals Litter Size Maintenance Mammalian Cell Massachusetts Mediating Methodology Methods Modeling Modification Mouse Strains Mus Mutation Operative Surgical Procedures Organism Ovum Pathway interactions Pregnancy Process Proteins Rattus Reagent Relative (related person)Reporting Research Research Personnel Respiratory System Rodent Sheep Surface System T-Lymphocyte TAP1 gene Techniques Technology Time Transgenic Mice Translating Whole Blood Zinc Fingers animal model development antigenic peptide transporter cost cost effective cystic fibrosis patients embryonic stem cell endonuclease human disease in vivo male mammalian genome mouse genome novel strategies nuclease pig genome pup recombinase repaired research study sperm cell synthetic protein technique development zygote

项目摘要

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.

描述（由申请人提供）：拟议研究的目的是开发新方法和/或采用当前可用的技术来对各种实验动物物种进行基因改造。目前，小鼠是研究中最常用的动物种类。它们的受欢迎程度源于其体型小、繁殖和饲养相对成本低、妊娠期短、窝产仔数大，以及大量的商业化试剂和技术。动物模型开发的最大进步之一是产生了表达外源 DNA 的小鼠（转基因小鼠、敲入小鼠）或删除了天然基因（敲除或基因缺陷小鼠）的小鼠技术。使用小鼠作为动物模型的一个主要缺点是它们通常无法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很难适应非啮齿动物物种，或者在其他物种中尝试成本高昂。人们已经开发了几种方法来克服这些障碍，最近的一些报告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 是一种合成蛋白，由与核酸内切酶融合的工程化 DNA 结合结构域组成，可诱导特定 DNA 序列中的双链断裂。 DNA 断裂通过宿主细胞修复途径进行修复，其中一部分修复包含导致基因突变的错误，实际上，这些错误通常会转化为目标基因产物的删除。这里描述的项目的总体目标是利用 ZFN 技术开发可靠且具有成本效益的方法来生产适用于跨物种的转基因动物。精子介导的基因转移（SMGT）将用于将 ZFN 传递至发育中的胚胎。使用这种方法，新鲜分离的精子将与编码 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运输。然后，携带 DNA 的精子可用于在体外或体内使卵子受精。这项技术将在两种物种（一种啮齿动物和一种非啮齿动物）中进行比较，以证明概念并产生转基因豚鼠和绵羊，它们是人类疾病的有价值的模型。该项目的具体目标是培育 1) 缺乏一种或多种参与免疫反应产生的基因的豚鼠，以及 2) 缺乏一种或多种参与心脏和呼吸系统胚胎发育的基因的绵羊。