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

基本信息

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
项目类别：
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

项目摘要

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）：大鼠是许多类型的人类疾病的偏爱模型，而小鼠不适合小鼠。与小鼠相反，大鼠和人类的细胞色素P450基因也具有更大的相似性，使大鼠成为毒理学和药理学研究的更有用的模型。大鼠也是糖尿病，关节炎，行为障碍（包括药物成瘾）和脑成像的最受欢迎的模型。然而，直到最近，由于缺乏能够促进生殖系的大鼠干细胞系以及缺乏修改基因组序列的有效技术，因此生成工程突变一直存在问题。转座原生物药物已经率先使用移动DNA元件（例如转座子）在大鼠生殖系中产生插入突变。迄今为止，我们已经制造了100多种插入突变线（称为TKOTM敲除大鼠模型）。这种方法利用基因陷阱策略来选择随机整合的转座子，从而可以快速识别序列标记的突变位点。在第一阶段的研究中，我们专注于从三个不同的转座子（SB），Piggybac（Pb）和TCBuster（TCB）的多动态转座酶合成，以提高细菌中换tosion的效率。我们报告了PB家族中成功产生多种多活跃转座酶。在第二阶段的研究中，我们将产生一个大鼠胚胎茎（RES）细胞库，其中包含超过200,000个双重报告基因陷阱插入突变。 EGFP报告基因系统将被用作Polya陷阱，以最大程度地提高大约30,000个大鼠基因中的每个大鼠基因中的插入突变的概率，以及无启动子TDTomato报告基因以筛选谱系特异性基因中断。从长远来看，我们打算使用RES Cell库在每个轨迹中生成大鼠敲除线。在第二阶段的研究中，我们将专注于在I阶段研究中创建的多活跃PB转座酶开发转座介导的敲除线，以产生一系列敲除线，这些敲除线将对多种应用，例如毒性，行为，行为和心脏病研究。我们将开发高通量的体外分化方案，以筛选以96孔格式培养的RES细胞池。通过筛选谱系特异性分化方案后包含TDTOMATO阳性细胞的井来筛选神经，心脏和内皮细胞谱系中的潜在突变。阳性井中的RES细胞池将被亚克隆并重新筛选，以识别带有潜在谱系特定突变的个体克隆。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侧翼DNA插入的序列。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，以排除影响无处不在表达基因座的插入。我们将为学术和药品最终用户开发两种产品。首先，我们将通过将这些细胞注入宿主胚泡中，从选定的RES细胞克隆中产生嵌合动物，并通过几轮反杂交将“无关”的突变隔离，以生成人类疾病的其他TKOTM敲除大鼠模型。其次，我们将销售Res Cell克隆，其中包含基因中的突变，这些突变超出了我们对学术和药物最终用户的核心利益。突变动物，从突变雄性中分离出的精子和突变的RES细胞将由国家大鼠资源和研究中心分布，并根据NIH政策与学术界共享，以共享用于生物医学研究的模型生物。公共卫生相关性：在应用“创建啮齿动物中诱变多动型转座酶的应用”中，我们正在寻求II期资金来使用我们创建的新型转座酶来生成新的人类疾病模型。我们已经证明了创建转座子介导的突变以建模人类疾病的基本和治疗研究应用程序的价值。我们以前的所有模型都是通过随机诱变获得的。在这项建议中，我们概述了将使我们能够鉴定出可能影响神经，心脏或血管功能的转座子介导的突变的研究。这样的大鼠模型将提供新的和有价值的工具，以开发在人类中特别普遍的疾病类类中的新疗法。因此，如果成功的话，该项目将通过使研究界很容易获得的人类疾病的大鼠突变产生大鼠的突变来受益。