Creation of hyperactive transposons for mutagenesis in rodents

创建用于啮齿动物诱变的高活性转座子

• 批准号: 8131644
• 负责人: ERIC M OSTERTAG
• 金额: $ 89.99万
• 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: 2010-09-01 至 2013-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8131644
• 关键词: Address; Affect; Animal Model; Animals; Arthritis; Backcrossings; Basic Science; Behavior Disorders; Behavioral; Biological Products; Biomedical Research; Blood Vessels; Brain imaging; Cardiac; Cardiovascular system; Cataloging; Catalogs; Cell Count; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell Lineage; Cell Separation; Cells; Characteristics; Code; Communities; Complement; Cryopreservation; Cytochrome P450; DNA; DNA Sequence; DNA Transposons; DNA analysis; Data; Defect; Derivation procedure; Diabetes Mellitus; Disease; Drug Addiction; Elements; Eligibility Determination; Embryo; Endothelial Cells; Engineering; Enhancers; Event; Exhibits; Family; Funding; Gene Expression; Generations; Genes; Genomics; Germ Lines; Goals; Heart; Hour; Housekeeping Gene; Human; In Situ; In Vitro; Individual; Knock-out; Lead; Libraries; Marketing; Mediating; Modeling; Molecular Profiling; Mus; Mutagenesis; Mutate; Mutation; Nature; Neurons; Noise; Pharmacologic Substance; Pharmacology and Toxicology; Phase; Phenotype; Plasmids; Policies; Population; Probability; Production; Protocols documentation; Public Health; Publishing; Quality Control; Rattus; Reporter; Reporter Genes; Reporting; Research; Resources; Rodent; Screening procedure; Seeds; Site; Sleeping Beauty; Sorting - Cell Movement; Southern Blotting; Stem cells; System; Technology; Therapeutic Human Experimentation; Tissues; Toxicology; Transfection; Transposase; United States National Institutes of Health; blastocyst; cell bank; controlled release; drug discovery; embryonic stem cell; falls; high throughput analysis; high throughput screening; human disease; in vivo; insight; interest; male; mutant; novel; phase 1 study; phase 2 study; public health relevance; rat genome; relating to nervous system; research study; sperm cell; stem; stem cell differentiation; stem cell technology; success; tool; transposon/insertion element; vector

DESCRIPTION (provided by applicant): The rat is a favored model for many types of human disease for which mice are not suitable. As opposed to the mouse, rats and humans also share more similarity in their cytochrome P450 genes, making the rat a more useful model for toxicology and pharmacology studies. The rat is also a favored model for diabetes, arthritis, behavioral disorders (including drug addiction), and brain imaging. However, until recently, generating engineered mutations has been problematic due to the lack of rat stem cell lines capable of contributing to the germ line, and the lack of efficient technologies to modify genomic sequences. Transposagen Biopharmaceuticals has pioneered the use of mobile DNA elements (e.g., transposons) to generate insertional mutations in the rat germ line. To date, we have over made over 100 insertional mutant lines (referred to as TKOTM Knockout Rat Models). This approach utilizes gene-trap strategies to select for randomly integrated transposons, which enable the rapid identification of sequence-tagged mutation sites. In Phase I studies, we focused on synthesizing hyperactive transposases, from three different families of transposons, Sleeping Beauty (SB), piggyBac (PB), and TcBuster (TcB), to increase the efficiency of transposition in the germ line. We report the successful generation of a number of hyperactive transposases in the PB family. In Phase II studies, we will generate a rat embryonic stem (rES) cell bank containing over 200,000 dual reporter gene-trap insertional mutations; a EGFP reporter system will be used as a polyA trap to maximize the probability of generating insertional mutations in each of the approximately 30,000 rat genes, and a promoterless tdTomato reporter to screen for lineage-specific gene disruptions. In the long term, we intend to use the rES cell bank to generate rat knockout lines in each locus. In Phase II studies, we will focus on developing transposon-mediated knockout lines in neural, cardiac, and endothelial cell lineages, using the hyperactive PB transposases created in Phase I studies, with the aim of generating a bank of knockout lines that that will be valuable for a wide variety of applications such as toxicology, behavioral, and cardiovascular research. We will develop high-throughput in vitro differentiation protocols to screen pools of rES cells cultured in 96-well formats. Potential mutations in neural, cardiac, and endothelial cell lineages will be identified by screening for wells that contain tdTomato positive cells after lineage-specific differentiation protocols. rES cell pools in positive wells will be subcloned and re-screened to identify the individual clone that carries the potential lineage specific mutation. Genomic DNA will be isolated and used as template for splinkerette PCR, which is used routinely to amplify sequences that flank DNA insertions. We will determine the genomic sites for each insertion and screen each gene for lineage-specific expression to rule out insertions that affect ubiquitously expressed loci. We will develop two products for academic and pharmaceutical end users. First, we will generate chimeric animals from selected rES cell clones by injecting these cells into host blastocysts, and segregate away the "irrelevant" mutations by several rounds of backcrosses to generate additional TKOTM Knockout Rat Models for human diseases. Second, we will market rES cell clones, containing mutations in genes that are beyond our core interests to academic and pharmaceutical end users. Mutant animals, sperm isolated from mutant males, and mutant rES cells will be distributed by the National Rat Resource and Research Center and shared with the academic community according to NIH policies for sharing model organisms for biomedical research. PUBLIC HEALTH RELEVANCE: In the application "Creation of hyperactive transposases for mutagenesis in rodents," we are seeking Phase II funding to use the novel transposases we created to generate new models of human disease. We have demonstrated the value of creating transposon-mediated mutations to model human diseases for basic and therapeutic research application. All of our previous models were obtained by random mutagenesis. In this proposal we outline studies that will enable us to identify transposon mediated mutations that will likely affect neural, heart, or blood vessel function. Such rat models will provide new and valuable tools to develop new therapies in classes of diseases that are particularly prevalent in humans. Thus, if successful, this project would benefit many goals of public health by making the production of mutations in the rat that model human diseases readily accessible to the research community.

描述（由申请人提供）：对于小鼠不适合的许多类型的人类疾病，大鼠是优选的模型。与小鼠不同，大鼠和人类的细胞色素 P450 基因也有更多相似性，这使得大鼠成为毒理学和药理学研究更有用的模型。大鼠也是糖尿病、关节炎、行为障碍（包括毒瘾）和脑成像的首选模型。然而，直到最近，由于缺乏能够形成种系的大鼠干细胞系，并且缺乏修饰基因组序列的有效技术，产生工程突变一直是个问题。 Transposagen Biopharmaceuticals 率先使用移动 DNA 元件（例如转座子）在大鼠种系中产生插入突变。迄今为止，我们已经制作了100多个插入突变株系（称为TKOTM敲除大鼠模型）。该方法利用基因陷阱策略来选择随机整合的转座子，从而能够快速识别序列标记的突变位点。在第一阶段研究中，我们专注于合成来自三个不同转座子家族：睡美人 (SB)、piggyBac (PB) 和 TcBuster (TcB) 的高活性转座酶，以提高种系中的转座效率。我们报告了 PB 家族中许多高活性转座酶的成功产生。在 II 期研究中，我们将生成一个包含超过 200,000 个双报告基因陷阱插入突变的大鼠胚胎干 (rES) 细胞库； EGFP报告系统将用作polyA陷阱，以最大限度地提高在大约30,000个大鼠基因中每个基因中产生插入突变的可能性，以及无启动子的tdTomato报告系统，以筛选谱系特异性基因破坏。从长远来看，我们打算利用 rES 细胞库在每个位点生成大鼠敲除系。在第二阶段研究中，我们将重点使用第一阶段研究中创建的高活性PB转座酶，在神经、心脏和内皮细胞谱系中开发转座子介导的敲除系，目的是生成一系列敲除系，这些敲除系对毒理学、行为和心血管研究等多种应用具有价值。我们将开发高通量体外分化方案来筛选以 96 孔形式培养的 rES 细胞库。神经细胞、心脏细胞和内皮细胞谱系的潜在突变将通过在谱系特异性分化方案后筛选含有 tdTomato 阳性细胞的孔来鉴定。阳性孔中的 rES 细胞池将被亚克隆并重新筛选，以鉴定携带潜在谱系特异性突变的单个克隆。基因组 DNA 将被分离并用作 splinkerette PCR 的模板，该模板通常用于扩增 DNA 插入侧翼的序列。我们将确定每个插入的基因组位点，并筛选每个基因的谱系特异性表达，以排除影响普遍表达位点的插入。我们将为学术和制药最终用户开发两种产品。首先，我们将通过将选定的 rES 细胞克隆注射到宿主囊胚中，从这些细胞中产生嵌合动物，并通过几轮回交分离出“不相关”的突变，以产生用于人类疾病的额外 TKOTM 敲除大鼠模型。其次，我们将向学术和制药最终用户推销包含超出我们核心利益的基因突变的 rES 细胞克隆。突变动物、从突变雄性中分离出的精子以及突变rES细胞将由国家大鼠资源和研究中心分发，并根据NIH生物医学研究共享模式生物的政策与学术界共享。 公共健康相关性：在“创建用于啮齿动物诱变的高活性转座酶”申请中，我们正在寻求第二阶段资金，以使用我们创建的新型转座酶来生成人类疾病的新模型。我们已经证明了创建转座子介导的突变来模拟人类疾病的价值，以用于基础和治疗研究应用。我们以前的所有模型都是通过随机诱变获得的。在本提案中，我们概述了一些研究，这些研究将使我们能够识别可能影响神经、心脏或血管功能的转座子介导的突变。这种大鼠模型将为开发针对人类特别普遍的疾病类别的新疗法提供新的和有价值的工具。因此，如果成功的话，该项目将通过使研究界能够轻松地在大鼠中产生模拟人类疾病的突变，从而有利于公共卫生的许多目标。