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: 7912098
• 负责人: ERIC M OSTERTAG
• 金额: $ 77.99万
• 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 2012-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912098
• 关键词: Address; Affect; Animal Model; Animals; Arthritis; Backcrossings; Behavior Disorders; Behavioral; 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)：大鼠是许多人类疾病的理想模型，而小鼠不适合这些疾病。与小鼠相反，大鼠和人类在细胞色素P450基因上也有更多的相似之处，使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(包括药物成瘾)和脑成像的理想模型。然而，直到最近，由于缺乏能够对生殖系做出贡献的大鼠干细胞系，以及缺乏修改基因组序列的有效技术，产生工程突变一直是有问题的。转基因生物制药公司率先使用可移动DNA元件(例如转座子)在大鼠生殖系中产生插入突变。到目前为止，我们已经制作了100多个插入突变系(称为TKOTM基因敲除大鼠模型)。这种方法利用基因捕获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。在第一阶段的研究中，我们专注于从三个不同的转座子家族，睡美人(SB)，猪Bac(PB)和TcBuster(TCB)合成高活性转座酶，以提高转座子在生殖系中的效率。我们报道了在PB家族中成功地产生了一些高活性转座酶。在第二阶段的研究中，我们将建立一个包含200,000多个双报告基因陷阱插入突变的大鼠胚胎干细胞(RES)细胞库；一个EGFP报告系统将被用作Polya陷阱，以最大限度地提高在大约30,000个大鼠基因中的每一个产生插入突变的可能性；以及一个无启动子的tdTomato报告系统，用于筛选特定血统的基因中断。从长远来看，我们打算利用RES细胞库在每个基因座上产生大鼠基因敲除系。在第二阶段研究中，我们将专注于利用第一阶段研究中产生的高活性PB转座酶，在神经、心脏和血管内皮细胞系中开发转座子介导的基因敲除系，目的是产生一组对毒理学、行为学和心血管研究等广泛应用有价值的基因敲除系。我们将开发高通量的体外分化方案来筛选以96孔格式培养的RES细胞池。神经、心脏和内皮细胞谱系的潜在突变将在谱系特异性分化方案后通过筛选含有tdTomato阳性细胞的孔来识别。阳性井中的RES细胞库将被亚克隆并重新筛选，以确定携带潜在谱系特异性突变的个体克隆。基因组DNA将被提取并用作模板用于夹板聚合酶链式反应，该模板通常用于扩增DNA插入片段两侧的序列。我们将确定每个插入的基因组位置，并筛选每个基因的谱系特异性表达，以排除影响普遍表达的基因座的插入。我们将为学术和制药终端用户开发两款产品。首先，我们将通过将选定的RES细胞克隆注入宿主囊胚来产生嵌合体动物，并通过几轮回交分离出“无关”的突变，以产生其他用于人类疾病的TKOTM基因敲除大鼠模型。其次，我们将向学术和制药终端用户销售RES细胞克隆，这些克隆包含超出我们核心利益的基因突变。突变动物、突变雄性精子和突变RES细胞将由国家老鼠资源和研究中心分发，并根据NIH关于共享生物医学研究模式生物的政策与学术界共享。 公共卫生相关性：在应用“在啮齿类动物中创建高活性转座酶用于突变”中，我们正在寻求第二阶段的资金，以使用我们创建的新型转座酶来产生新的人类疾病模型。我们已经证明了创造转座子介导的突变来为基础和治疗研究应用建立人类疾病模型的价值。我们以前的所有模型都是通过随机诱变获得的。在这项提案中，我们概述了一些研究，这些研究将使我们能够识别转座子介导的突变，这些突变可能会影响神经、心脏或血管功能。这种大鼠模型将提供新的、有价值的工具，以开发针对人类特别流行的疾病的新疗法。因此，如果成功，该项目将使研究团体能够容易地获得模拟人类疾病的大鼠突变，从而使公共卫生的许多目标受益。