Creation of hyperactive transposons for mutagenesis in rodents

描述（由申请人提供）：大鼠是许多小鼠不适合的人类疾病的首选模型。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，这使得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）和脑成像的首选模型。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，产生工程突变一直存在问题。转座子生物制药公司率先使用可移动的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，我们已经制造了超过100个插入突变系（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I期研究中，我们专注于合成来自三个不同转座子家族的高活性转座子，睡美人（SB）， piggyBac （PB）和TcBuster (TcB)，以提高种系转座子的效率。我们报道了PB家族中一些过度活跃转座的成功产生。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诱捕器，以最大限度地提高在大约30,000个大鼠基因中产生插入突变的可能性，而无启动子的tdTomato报告系统将用于筛选谱系特异性基因破坏。从长远来看，我们打算使用rES细胞库在每个位点产生大鼠敲除系。在II期研究中，我们将重点开发转座子介导的神经、心脏和内皮细胞系的基因敲除系，使用I期研究中产生的超活性PB转座酶，目的是产生一系列基因敲除系，这些基因敲除系将在毒理学、行为学和心血管研究等广泛应用中具有价值。我们将开发高通量体外分化方案，筛选96孔格式培养的rES细胞池。神经、心脏和内皮细胞系的潜在突变将通过筛选含有tdTomato阳性细胞的细胞孔，在谱系特异性分化方案后进行鉴定。将对阳性井中的rES细胞池进行亚克隆并重新筛选，以确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，splinkerette PCR通常用于扩增DNA插入侧的序列。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终端用户开发两种产品。首先，我们将从选定的rES细胞克隆中注入这些细胞到宿主囊胚中，产生嵌合动物，并通过几轮回交分离“无关”突变，以产生用于人类疾病的额外的TKOTM敲除大鼠模型。其次，我们将向学术和制药最终用户推销含有超出我们核心兴趣的基因突变的rES细胞克隆。突变动物、从突变雄性中分离出的精子和突变的rES细胞将由国立大鼠资源研究中心分发，并根据NIH的生物医学研究模式生物共享政策与学术界共享。

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.