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Development of novel rat models for site-specific transgene integration

开发用于位点特异性转基因整合的新型大鼠模型

基本信息

批准号：
8643473
负责人：
Ruby Yanru Chen-Tsai
金额：
$ 21.79万
依托单位：
APPLIED STEMCELL, INC.
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8643473
关键词：
Aging Animal Model Animals Autoimmunity Bacteriophages Biological Biological Models Biology Biomedical Research Bypass Cancer Model Cardiovascular Diseases Cells Communicable Diseases Communities Complex DNA Development Docking Embryo Ensure Event Gene Expression Gene Silencing Gene Targeting Generations Genes Genetic Processes Genetic Recombination Genetically Modified Animals Genomic Instability Genomics Goals Health Human Hypertension Inflammation Injection of therapeutic agent Integrase Intercistronic Region Knock-in Mouse Knock-out Laboratory Rat Life Malignant Neoplasms Mediating Medical Medical Research Methods Microinjections Mission Modeling Molecular Mus Nerve Degeneration Neurobiology Pathologic Processes Pharmacology and Toxicology Physiology Plasmid Cloning Vector Plasmids Positioning Attribute Production Rat Strains Rattus Reagent Reporter Resources Retinitis Pigmentosa Risk Assessment Site Stem cells Study models System Technology Time Transcription Coactivator Transgenes Transgenic Mice Transgenic Organisms Transplantation United States National Institutes of Health Zinc Fingers addiction base behavioral pharmacology burden of illness cancer risk commercialization cost cost effective disability embryonic stem cell empowered genetic element genetic manipulation glomerulosclerosis homologous recombination human disease induced pluripotent stem cell insight interest mouse model novel nuclease offspring public health relevance rat genome site-specific integration tool transgene expression vector zinc finger nuclease zygote

项目摘要

The goal of this proposal is to develop novel rat strains that can be used to efficiently generate site-specific transgenic rat models via pronuclear microinjection. The laboratory rat (R. norvegicus) is a central experimental animal in several fields of biomedical research, such as cardiovascular diseases, aging, infectious diseases, autoimmunity, cancer models, transplantation biology, inflammation, cancer risk assessment, industrial toxicology, pharmacology, behavioral and addiction studies, and neurobiology. Up till recently, the ability of creating genetically modified rats has been limited compared to that in the mouse mainly due to lack of genetic manipulation tools and technologies in the rat. The isolation and establishment of rat embryonic stem (rES) cells by Drs. Ying and Smith's groups at the end of 2008 revolutionized the capability of making genetically modified rat models. As a result, rat transgenics is becoming as precise and powerful as has been the case in mice, yielding better models of human diseases. Recent advances in nucleases such as Zinc-finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs) have been successfully used to construct 'knockout' rat models by injecting gene targeting molecular complexes directly into an embryo for chimeric offspring production, avoiding the need to use any type of stem cells. To facilitate the generation and use of rat models of human diseases, it is critical to develop systems that enable fast, efficient and precise introduction of exogenous genetic elements into the rat genome. Here, we propose to use the bacteriophage integrase system to direct transegene integration at transcriptionally active genomic loci with higher integration efficiency. Integrases such as phiC31 or Bxb1 carries out efficient, unidirectional recombination between two non-identical sites, attP and attB. In this proposal, we will identify transcriptionally active loci in the rat genome and insert attP sites at such loci using TALEN-mediated homologous recombination (HR). These attP-containing rats will be used as embryo donors for pronuclear injection of the transgene on an attB plasmid. In the presence of integrases, recombination between attP and attB results in an insertion of the transgene precisely at the attP site in the rat genome. This technology will allow a fast, efficient generation of knockin rat models containing any gene of interest with consistent, stable, guaranteed gene expression. Advantages of this integrase-based technology are: (1) Transgene integration happens at pre-selected and transcriptionally active loci; (2) Site-specific knockin rat models are made by direct injection of the DNA into the rat zygotes, bypassing rES cells; (3) Gene integration efficiency is much higher, but off-target events are lower compared to ZFNs or TALENs. Successful execution of this project will create a cost-effective method and valuable resources for the bio-medical community who employ rat models for their studies of human diseases. ! !

这项提案的目标是开发新的大鼠品系，可用于有效地产生通过原核显微注射建立位点特异性转基因大鼠模型。实验室大鼠（R. norvegicus）是一种生物医学研究的几个领域的中心实验动物，如心血管疾病，衰老、传染病、自身免疫、癌症模型、移植生物学、炎症、癌症风险评估、工业毒理学、药理学、行为和成瘾研究，以及神经生物学到目前为止，与此相比，创造转基因老鼠的能力受到限制。这主要是由于缺乏基因操作工具和技术。隔离 2008年底，Ying博士和Smith博士的团队建立了大鼠胚胎干细胞（rES）彻底改变了制作转基因老鼠模型的能力。因此，老鼠转基因是变得像老鼠一样精确和强大，产生了更好的人类模型。疾病核酸酶如锌指核酸酶（ZFN）和转录的最新进展激活物样效应物核酸酶（TALEN）已成功地用于构建"敲除"大鼠通过将基因靶向分子复合物直接注射到胚胎中以产生嵌合后代的模型生产，避免需要使用任何类型的干细胞。为了促进人类疾病的大鼠模型的产生和使用，能够快速、有效和精确地将外源遗传元件引入大鼠的系统基因组在这里，我们建议使用噬菌体整合酶系统来指导转基因整合，转录活性基因座具有更高的整合效率。整合酶如phiC31或Bxb1 在两个不同的位点attP和attB之间进行有效的单向重组。在这我们将在大鼠基因组中鉴定转录活性位点，并在这些位点上插入attP位点使用TALEN介导的同源重组（HR）。这些含有attP的大鼠将被用作用于attB质粒上的转基因的原核注射的胚胎供体。存在下在整合酶的作用下，attP和attB之间的重组导致转基因精确地插入到整合酶的下游。 attP位点。这项技术将允许快速，有效地产生敲入大鼠模型包含任何感兴趣的基因，具有一致的、稳定的、有保证的基因表达。等优点基于整合酶技术是：（1）转基因整合发生在预先选择的和转录的活性位点;（2）通过将DNA直接注射到大鼠体内来制备位点特异性敲入大鼠模型（3）基因整合效率高得多，但脱靶事件发生率低。与ZFN或TALEN相比更低。该项目的成功实施将创造一个具有成本效益的方法和宝贵的资源，为生物医学界谁采用大鼠模型的研究，人类疾病。 ! !