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: 7911506
• 负责人: ERIC M OSTERTAG
• 金额: $ 18.02万
• 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 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7911506
• 关键词: 4-Hydroxy-Tamoxifen; Adult; Alkaline Phosphatase; Alleles; Animal Model; Animals; Biological; Biology; Breeding; Cancer Biology; Cells; Chimeric Proteins; Cloning; Communities; DNA Transposons; Development; Disease; 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）： 目的是通过体内基因陷阱中介导的转座子介导的特异性重组酶（SSR）表达转基因大鼠。大鼠与建模人类生物学和疾病非常相关，因为与小鼠不同，大鼠与人具有关键的生物学相似性。虽然体外基因诱捕诱变可用于基因发现有用，但具有两个关键原因是一种快速而有效的体内方法：1）体内基因陷阱将揭示正宗的时空表达模式，而2）则可以立即将这些遗传资源用于大鼠中的基因组操纵。转基因动物中的分散启动子分析显然不足以快速和经济地生成用于SSR介导的组织特异性基因组操纵的有效工具。转基因大鼠的产生实在太贵了，并且仅在几个机构进行。小鼠启动子元素可用于产生表达SSR的转基因大鼠，但是大多数启动子元素缺乏关键调节序列，对基因组插入位点的位置敏感，或者不一定会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成千上万的转录元素，因此需要一种有效的体内方法来真实，快速地概括内源性表达模式，以生成各种有效的转基因工具。此外，鉴定新抄录元素的一种方法将加速对哺乳动物基因组学的全面理解。 基因陷阱诱变是一种标准方法，用于识别和利用新的转录单元，但是对于快速有效的体内筛选，传统策略（例如针对胚胎干细胞的靶向）根本无法实现。通过使用我们在Transposagen增强的PiggyBac（PB）转座子系统，我们将追求大鼠体内启动子陷阱。使用明亮的荧光蛋白（FPS），将在活胚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荧光都标志着每个被困元件的表达结构域。 CRE-EGFP盒式磁带由PB转座子传递，该PB转座子以一个转基因系列（称为“供体”）为转基因助剂。另一个转基因线“驱动器”提供了PB转座酶在种系中的表达。转座子动员只是由杂交驱动器和供体系引发的，因此PB转座酶在双转基因动物中动员了Cre-EGFP基因诱捕基因陷阱转座子，称为“种子”大鼠。 Pb是迄今为止在哺乳动物细胞中基因诱变的最有效的转座子，通过使用增强的Pb转座酶，我们期望每个配子的插入速率至少应产生一个基因陷阱事件。结果，从种子大鼠繁殖的每个G1后代都会显示出Cre-EGFP记者的独特表达模式。由于它是插入诱变者，因此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荧光将记录在E13.5和E16.5胚胎中，并且插入位点的鉴定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。加速大鼠遗传操作工具的策略对于我们在这里的方法是不可或缺的。通过使用CRE-EGFP记者，我们将创建重组酶介导的有条件诱变的工具。为了监测CRE活性，我们将创建转基因大鼠在CRE重组事件后在细胞中表达TDTOMATO荧光蛋白。 TDTOMATO蛋白表现出非常明亮的荧光，已证明对体内表达分析有用。使用驱动普遍表达的大鼠Rosa26启动子，仅在CRE介导的中间序列去除后，TDTOMATO表达才能激活。这些转基因大鼠将与上述从种子大鼠获得的G1后代配对。我们将分析EGFP和TDTOMATO荧光的Live G2胚胎。通过使用这些FPS，可以实时监测每个基因陷阱。这将为未来的调查中进一步进行深入分析创造机会。我们将在本屏幕中至少在活胚中确定至少25个独特的杂物，作为概念证明。 除TDTOMATO外，CRE重组酶活性还将触发FLPERT2表达，该表达与TDTOMATO开放式阅读框架相关联，通过内部核糖体表达序列。 FLPERT2活性取决于4-羟基莫昔芬（4-OHT），因此提供了可诱导的FLP重组酶活性。我们的设计启用了两个关键的FLP介导的操作：1）FLP重组酶诱导（通过4-OHT）通过侧翼FLP重物组合酶靶位点和2）FLPERT2表达将启用FLP依赖性依赖FLP的FRT辅助等位基因（通过其他努力生成）。 这里生成的工具和资源将为大鼠遗传学提供巨大的进步。该屏幕将至少识别25个转录元素，这些元素表现出Cre-EGFP的独特体内表达模式。我们的策略将为轻松生成数百种其他基因陷阱线提供机会。我们的研究将在未来的研究中为重组酶介导的条件诱变产生有价值的大鼠转基因线。 公共卫生相关性： 该建议描述了一种快速发现和利用大鼠基因组中各种基因的组织特异性表达模式的力量（其中打开的基因）。使用移动DNA元件或称为PiggyBac的“跳跃基因”，我们将在活大鼠胚胎中标记数百个基因。然后，在未来的研究中将利用显示的独特表达模式作为组织特异性诱变的工具。大鼠高度适合寻求对人类遗传学和生物学的更好理解，因为与其他流行的动物模型（如小鼠）相比，大鼠在许多方面都更好地类似于人类病理学，生理学，神经病学和癌症生物学。这项研究的结果将生成有价值的遗传工具，以对特定组织中的基因功能进行精致和微妙的研究。