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: 8330384
• 负责人: ERIC M OSTERTAG
• 金额: $ 4.2万
• 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/8330384
• 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; Health; 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; 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.

描述（由申请人提供）： 我们的目标是通过转座子介导的体内基因诱捕，以有效和无偏见的方式建立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，因为大鼠与小鼠不同，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是有用的，但是快速且有效的体内方法是优选的，这有两个关键原因：1）体内基因陷阱将揭示真实的时空表达模式，以及2）这些遗传资源然后可以立即用于大鼠中的基因组操作。在转基因动物中的片段启动子分析显然不足以快速和经济地产生用于SSR介导的组织特异性基因组操作的有效工具。转基因大鼠的生产成本太高，而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万个转录元件，需要一种有效的体内方法来真实且快速地重现内源性表达模式以产生多样化且有效的转基因工具集。此外，鉴定新转录元件的方法将加速对哺乳动物基因组学的全面理解。基因陷阱诱变是鉴定和开发新转录单位的标准方法，但传统的策略，如靶向胚胎干细胞的策略，对于快速有效的体内筛选来说是站不住脚的。通过使用我们在Transposagen增强的PiggyBac（PB）转座子系统，我们将在大鼠中追求体内启动子陷阱。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。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荧光标记每个捕获元件的表达结构域。Cre-EGFP盒陷阱由PB转座子递送，所述PB转座子在一个转基因系（称为“供体”）中起源为转基因多联体。另一个转基因株系，“驱动株”，提供PB转座酶在生殖株系中的表达。转座子动员是简单地启动杂交驱动和供体线，这样PB转座酶动员Cre-EGFP基因陷阱转座子在生殖细胞内的双转基因动物，称为“种子”大鼠。PB是迄今为止描述的用于哺乳动物细胞中基因诱变的最有效的转座子，并且通过使用增强的PB转座酶，我们期望插入率应该产生每个配子至少一个基因陷阱事件。因此，从种子大鼠繁殖的每个G1后代将显示Cre-EGFP报告基因的独特表达模式。因为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。一种加速大鼠遗传操作工具的策略是我们这里方法的组成部分。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，我们将创建在Cre重组事件后在细胞中表达tdTomato荧光蛋白的转基因大鼠。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ROSA 26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。我们将分析活的G2胚胎的EGFP和tdTomato荧光。通过使用这些FP，可以实时监测每个基因陷阱;这将为未来的研究提供进一步深入分析的机会。我们将在活胚胎中的该筛选中鉴定至少25个独特的Cre表达元件，作为概念证明。除了tdTomato之外，Cre重组酶活性还将触发FlpERT 2表达，其通过内部核糖体表达序列与tdTomato开放阅读框连接。FlpERT 2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介导的操作：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，以及2）FlpERT 2表达将实现含FRT等位基因的Flp依赖性条件修饰（通过其他努力产生）。这里产生的工具和资源将为大鼠遗传学提供巨大的进步。该筛选将鉴定出至少25种表现出独特的Cre-EGFP体内表达模式的转录元件。我们的策略将提供容易地产生数百个另外的基因捕获系的机会。我们的研究将产生有价值的大鼠转基因株系重组酶介导的条件诱变在未来的研究。