Development of novel rat models for site-specific transgene integration

这项提议的目标是开发新的老鼠品系，这种品系可以用来高效地产生 原核显微注射定位转基因大鼠模型。实验鼠(褐家鼠)是一种 几个生物医学研究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。 直到最近，创造转基因大鼠的能力与之相比一直是有限的 在小鼠中，主要是由于缺乏遗传操作工具和技术在大鼠中。这种隔绝 并于2008年底由Ying和Smith博士的团队建立了大鼠胚胎干细胞(RES) 彻底改变了制造转基因大鼠模型的能力。因此，老鼠转基因技术 变得像老鼠一样精确和强大，产生了更好的人类模型 疾病。锌指核酸酶等核酸酶及其转录的研究进展 激活子样效应子核酸酶(TALEN)已成功地用于构建“基因敲除”大鼠 将基因靶向分子复合体直接注射到嵌合子代胚胎中的模型 生产，避免使用任何类型的干细胞。 为了促进人类疾病大鼠模型的产生和使用，关键是开发 能够快速、高效和精确地将外源基因元素导入大鼠的系统 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较高的整合效率。整合酶，如phiC31或Bxb1 在两个不相同的站点attP和attB之间执行高效的单向重组。在这 建议，我们将在大鼠基因组中识别转录活性基因座并在这些基因座上插入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 将转基因基因原核注射到attB质粒上的胚胎供体。在……面前 整合酶，attP和attB之间的重组导致转基因恰好插入到 大鼠基因组中的attP位点。这项技术将允许快速、高效地产生敲门大鼠模型 包含具有一致、稳定、有保证的基因表达的任何感兴趣的基因。这样做的好处 基于整合酶的技术是：(1)转基因整合发生在预先选择的和转录的 (2)直接将DNA注入大鼠体内，建立定位敲打大鼠模型 合子，绕过RES细胞；(3)基因整合效率高得多，但脱靶事件 低于ZFN或TALEN。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，为生物医学界谁使用大鼠模型的研究 人类疾病。 好了！ 好了！

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. ! !