Generation of Site-Specific Recombinase-Expressing Transgenic Rats using an Enhan

使用 Enhan 生成表达位点特异性重组酶的转基因大鼠

• 批准号: 8139285
• 负责人: ERIC M OSTERTAG
• 金额: $ 6.05万
• 依托单位: TRANSPOSAGEN BIOPHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-09 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8139285
• 关键词: 4-Hydroxy-Tamoxifen; Adult; Alkaline Phosphatase; Alleles; Animal Model; Animals; Biological; Breeding; Cancer Biology; Cells; Chimeric Proteins; Cloning; Communities; DNA Transposons; Development; Drug Industry; Elements; Embryo; Event; Excision; Exhibits; Fluorescence; Future; Gene Delivery; Generations; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Genomics; Germ Cells; Germ Lines; Germ-Line Mutation; Goals; Human; Human Biology; Human Genetics; Human Pathology; Humulus; In Vitro; Indium; Individual; Inherited; Institution; Introns; Investigation; Jumping Genes; Knock-out; Lead; Life; Link; Location; Mammalian Cell; Mediating; Methods; Modeling; Modification; Monitor; Mus; Mutagenesis; Mutagens; Mutation; Neurology; Open Reading Frames; Organism; Partner in relationship; Pattern; Phase; Physiology; Positioning Attribute; Production; Proteins; Rattus; Reporter; Resources; Seeds; Site; Sleeping Beauty; Stem cells; System; Techniques; Technology; Time; Tissues; Toxicology; Transgenes; Transgenic Animals; Transgenic Organisms; Transposase; Validation; design; embryonic stem cell; falls; fusion gene; gene discovery; gene function; genetic manipulation; genetic resource; homologous recombination; human disease; in vivo; mammalian genome; mouse model; mutant; offspring; promoter; public health relevance; rapid technique; rat genome; recombinase; red fluorescent protein; retroviral transduction; sperm cell; success; tool; transposon/insertion element; vertebrate genome

DESCRIPTION (provided by applicant): The goal is to create site-specific recombinase (SSR)-expressing transgenic rats in an efficient and unbiased manner through a transposon-mediated in vivo gene trap. Rats are very relevant for modeling human biology and disease because rats, unlike mice, possess key biological similarities to humans. While in vitro gene trap mutagenesis is useful for gene discovery, a rapid and efficient in vivo method is preferable for two key reasons: 1) an in vivo gene trap will reveal an authentic spatio-temporal expression pattern, and 2) these genetic resources of can then be immediately employed for genome manipulations in the rat. Piecemeal promoter analysis in transgenic animals is clearly not sufficient for rapidly and economically generating effective tools for SSR-mediated tissue-specific genome manipulation. The generation of transgenic rats is simply too expensive, and is performed at only a few institutions. Mouse promoter elements could be used to generate SSR-expressing transgenic rats, but most promoter elements lack key regulatory sequences, are sensitive to position effects at genomic insertion sites, or will not necessarily produce identical expression patterns across species. Considering that tens of thousands of transcribed elements likely exist in the mammalian genome, an efficient in vivo method is needed to authentically and rapidly recapitulate endogenous expression patterns to generate a diverse and effective set of transgenic tools. Furthermore, a method to identify new transcribed elements would accelerate a comprehensive understanding of mammalian genomics. Gene trap mutagenesis is a standard approach for identifying and exploiting new transcription units, but traditional strategies, such as those targeting embryonic stem cells, are simply untenable for a rapid and efficient in vivo screen. By using a PiggyBac (PB) transposon system that we have enhanced at Transposagen, we will pursue an in vivo promoter trap in rats. Promoter traps will be visualized in live embryos and animals using bright fluorescent proteins (FPs). The primary reporter in this promoter trap is a Cre-EGFP fusion protein, in which both Cre recombinase activity and EGFP fluorescence marks the expression domain of each trapped element. The Cre-EGFP cassette trap is delivered by a PB transposon that originates as a transgene concatemer in one transgenic line, called the "donor." Another transgenic line, the "driver," provides expression of the PB transposase in the germ line. Transposon mobilization is simply initiated by interbreeding driver and donor lines, such that the PB transposase mobilizes the Cre-EGFP gene trap transposon within germ cells in double transgenic animals, designated as "seed" rats. PB is the most efficient transposon yet described for gene mutagenesis in mammalian cells, and by using an enhanced PB transposase, we expect an insertion rate that should yield at least one gene trap event per gamete. As a result, each G1 offspring bred from seed rats will display a unique expression pattern of the Cre-EGFP reporter. Because it is an insertional mutagen, the integration site of the PB transposon is easily determined by simple PCR cloning techniques. EGFP fluorescence will be documented in E13.5 and E16.5 embryos and the identification of insertion sites will enable one to link a specific expression pattern with specific genomic locations. As noted above, tools are needed for expanding the genetic resources of the rat. A strategy to accelerate the tools for genetic manipulations in the rat is integral to our approach here. By using a Cre-EGFP reporter, we will be creating recombinase-mediated tools for conditional mutagenesis. To monitor Cre activity we will create transgenic rats that express a tdTomato fluorescent protein in cells following a Cre recombination event. The tdTomato protein exhibits very bright fluorescence, and has proven useful for in vivo expression analysis. Using the rat ROSA26 promoter that drives ubiquitous expression, tdTomato expression will be activated only following Cre-mediated removal of an intervening sequence. These transgenic rats will be mated to the G1 offspring described above that are obtained from seed rats. We will analyze live G2 embryos for EGFP and tdTomato fluorescence. By using these FPs, each gene trap can be monitored in real-time; this will create the opportunity for a further in depth analyses in future investigations. We will identify at least 25 unique Cre- expressing elements in this screen in live embryos, as a proof of concept. In addition to tdTomato, Cre recombinase activity will also trigger FlpERT2 expression, which is linked to the tdTomato open reading frame via an internal ribosomal expression sequence. FlpERT2 activity is dependent on 4-hydroxytamoxifen (4-OHT), and thus provides inducible Flp recombinase activity. Our design enables two key Flp-mediated manipulations: 1) Flp recombinase induction (via 4-OHT) removes the Cre-EGFP gene trap through flanking Flp recombinase target sites and 2) FlpERT2 expression will enable Flp-dependent conditional modification of FRT-containing alleles (generated through other efforts). The tools and resources generated here will provide great advances for rat genetics. This screen will identify at least 25 transcribed elements exhibiting a unique in vivo expression pattern of Cre-EGFP. Our strategy will provide the opportunity to easily generate hundreds of additional gene trap lines. Our investigation will generate valuable rat transgenic lines for recombinase-mediated conditional mutagenesis in future studies. PUBLIC HEALTH RELEVANCE: This proposal describes a rapid method to discover and harness the power of tissue-specific expression patterns (where genes are turned "on") of a wide variety of genes in the rat genome, in live animals. Using a mobile DNA element, or "jumping gene," called piggyBac, we will tag hundreds of genes in live rat embryos. The unique expression patterns that are revealed will then be exploited as tools for tissue-specific mutagenesis in future studies. The rat is highly suitable for seeking a better understanding of human genetics and biology because the rat in many ways better resembles human pathology, physiology, neurology, and cancer biology than other popular animal models, such as the mouse. The results from this study will generate valuable genetic tools for refined and subtle investigations of gene function in specific tissues.

描述（由申请人提供）： 目标是通过转座子介导的体内基因陷阱，以高效且公正的方式创建表达位点特异性重组酶（SSR）的转基因大鼠。大鼠与人类生物学和疾病建模非常相关，因为与小鼠不同，大鼠与人类具有关键的生物学相似性。虽然体外基因陷阱诱变对于基因发现有用，但快速有效的体内方法更可取，原因有两个：1）体内基因陷阱将揭示真实的时空表达模式，2）这些遗传资源可以立即用于大鼠的基因组操作。转基因动物中的零碎启动子分析显然不足以快速且经济地生成用于 SSR 介导的组织特异性基因组操作的有效工具。转基因老鼠的产生成本太高，并且只有少数机构进行。小鼠启动子元件可用于产生表达SSR的转基因大鼠，但大多数启动子元件缺乏关键调控序列，对基因组插入位点的位置效应敏感，或者不一定在物种之间产生相同的表达模式。考虑到哺乳动物基因组中可能存在数以万计的转录元件，因此需要一种有效的体内方法来真实、快速地重现内源表达模式，以生成一组多样化且有效的转基因工具。此外，识别新转录元件的方法将加速对哺乳动物基因组学的全面了解。 基因陷阱诱变是识别和利用新转录单位的标准方法，但传统策略（例如针对胚胎干细胞的策略）对于快速有效的体内筛选来说根本站不住脚。通过使用我们在 Transposagen 增强的 PiggyBac (PB) 转座子系统，我们将在大鼠体内寻找启动子陷阱。将使用明亮的荧光蛋白 (FP) 在活胚胎和动物中可视化启动子陷阱。该启动子陷阱中的主要报告基因是 Cre-EGFP 融合蛋白，其中 Cre 重组酶活性和 EGFP 荧光都标记了每个捕获元件的表达域。 Cre-EGFP 盒式陷阱由 PB 转座子传递，该转座子起源于一个转基因品系（称为“供体”）中的转基因多联体。另一种转基因系“驱动器”在种系中提供PB转座酶的表达。转座子动员简单地通过杂交驱动系和供体系启动，使得PB转座酶在双转基因动物（称为“种子”大鼠）的生殖细胞内动员Cre-EGFP基因捕获转座子。 PB 是迄今为止在哺乳动物细胞中用于基因诱变的最有效的转座子，通过使用增强的 PB 转座酶，我们预计每个配子的插入率应产生至少一个基因捕获事件。因此，种子鼠培育出的每个 G1 后代都会表现出独特的 Cre-EGFP 报告基因表达模式。由于它是一种插入诱变剂，PB 转座子的整合位点很容易通过简单的 PCR 克隆技术确定。 EGFP 荧光将在 E13.5 和 E16.5 胚胎中记录，插入位点的识别将使人们能够将特定的表达模式与特定的基因组位置联系起来。 如上所述，需要工具来扩大大鼠的遗传资源。加速老鼠基因操作工具的策略是我们这里的方法的组成部分。通过使用 Cre-EGFP 报告基因，我们将创建重组酶介导的条件诱变工具。为了监测 Cre 活性，我们将创建转基因大鼠，在 Cre 重组事件后，它们在细胞中表达 tdTomato 荧光蛋白。 tdTomato 蛋白表现出非常明亮的荧光，并已被证明可用于体内表达分析。使用驱动普遍表达的大鼠 ROSA26 启动子，只有在 Cre 介导的插入序列去除后，tdTomato 表达才会被激活。这些转基因大鼠将与上述从种子大鼠获得的 G1 后代交配。我们将分析活 G2 胚胎的 EGFP 和 tdTomato 荧光。通过使用这些FP，可以实时监控每个基因陷阱；这将为未来调查中进一步深入分析创造机会。我们将在活胚胎的筛选中鉴定出至少 25 个独特的 Cre 表达元件，作为概念证明。 除了 tdTomato 之外，Cre 重组酶活性也会触发 FlpERT2 表达，该表达通过内部核糖体表达序列与 tdTomato 开放阅读框相连。 FlpERT2 活性依赖于 4-羟基他莫昔芬 (4-OHT)，因此提供诱导型 Flp 重组酶活性。我们的设计实现了两个关键的 Flp 介导操作：1) Flp 重组酶诱导（通过 4-OHT）通过侧翼 Flp 重组酶靶位点去除 Cre-EGFP 基因陷阱，2) FlpERT2 表达将使包含 FRT 的等位基因（通过其他努力产生）实现 Flp 依赖性条件修饰。 这里产生的工具和资源将为大鼠遗传学提供巨大进步。该筛选将鉴定至少 25 个表现出 Cre-EGFP 独特体内表达模式的转录元件。我们的策略将提供轻松生成数百个额外基因陷阱系的机会。我们的研究将在未来的研究中产生有价值的大鼠转基因系，用于重组酶介导的条件诱变。 公共卫生相关性： 该提案描述了一种快速方法来发现和利用活体动物中大鼠基因组中多种基因的组织特异性表达模式（其中基因“打开”）的力量。使用称为piggyBac的移动DNA元件或“跳跃基因”，我们将标记活体大鼠胚胎中的数百个基因。所揭示的独特表达模式将在未来的研究中用作组织特异性诱变的工具。大鼠非常适合寻求更好地了解人类遗传学和生物学，因为大鼠在许多方面比其他流行的动物模型（例如小鼠）更类似于人类病理学、生理学、神经学和癌症生物学。这项研究的结果将为特定组织中基因功能的精细和微妙研究提供有价值的遗传工具。