Transposon mutagenesis for modeling complex human disease in rats

描述(由申请人提供)：理解复杂人类疾病的遗传基础的关键限制之一是良好模型的可用性。实验大鼠就是这样一种模型，由于深入研究了与人类在生理、病理和序列上的相似性，它具有巨大的价值。在大鼠的遗传学研究中，已经定位了1300多个与疾病相关的数量性状基因座(QTL)。然而，在老鼠研究领域，生理学和遗传学之间存在着脱节，因为没有有效的方法来突变和识别基因。我们一直在开发一种新工具来缩小这一技术差距。我们现在正在建立的初步数据显示，使用睡美人(SB)转座子系统的转座子插入突变可以在大鼠中产生可遗传突变，其效率是传统方法的50倍。我们相信，SB转座的特性，特别是转座子跳到染色体内邻近序列的趋势(局部跳跃)，将使我们能够针对这些区域进行突变，并显著加快疾病基因的发现。转座子突变的另一个强大应用是发现癌症基因。在转基因啮齿动物的体细胞中，SB转座子插入突变可以通过罕见但重复的癌症基因突变来驱动肿瘤的形成。我们可以使用转座子通过研究它们在肿瘤DNA中的插入位置来寻找这些癌症基因。我们特别建议通过1)开发多个在基因组不同区域具有转座子的转基因菌株来利用局部跳跃在QTL区域内进行转座子突变，以及2)开发SB转座子系统用于大鼠的体细胞突变，从而并行地开发这两个应用来模拟遗传病。我们选择Dahl盐敏感(SS)近交系大鼠作为我们的模型，是因为它在众多研究人员中处于心血管研究的前沿，我们初步认为它是一种新的人类乳腺癌模型。我们将SS菌株和SB转座子突变与数十年的生理和病理特征相结合，建立疾病模型，并发现相关的疾病基因。叙述：研究人员开发心血管疾病和癌症等复杂人类疾病的诊断测试和治疗方法的关键是找到相关基因。我们使用我们所称的‘遗传模式生物’，如老鼠，来了解不同疾病的病理，并努力发现相关基因，通过突变或移除它们，并确定发生了什么。我们正在开发一种强大的新工具，它在老鼠身上做这件事时要高效得多，这样我们就可以加快在许多疾病领域的研究，以提高我们所有人的生活质量。

DESCRIPTION (provided by applicant): One of the key limitations to understanding the genetic basis of complex human disease is the availability of good models. The laboratory rat is one such model which has tremendous value because of intensely studied physiological, pathological and sequence similarities to humans. More than 1300 disease-related quantitative trait loci (QTL) have been mapped in genetic studies in the rat. There is a disconnection, however, in the rat research field between physiology and genetics because there are no efficient means to mutate and identify genes. We have been developing a new tool to close this technology gap. We are now building upon preliminary data showing that transposon insertional mutagenesis using the Sleeping Beauty (SB) transposon system can generate heritable mutations in the rat at a rate which is 50-fold more efficient than conventional methods. We believe that properties of SB transposition, specifically the tendency for transposons to jump to nearby sequences within a chromosome (local hopping), will allow us to target these regions for mutagenesis and dramatically accelerate disease gene discovery. Another powerful application of transposon mutagenesis is cancer gene discovery. In somatic cells of transgenic rodents, SB transposon insertional mutagenesis can drive tumor formation by the rare, but repeated mutation of cancer genes. We can use the transposon to find these cancer genes by studying their insertion sites in tumor DNA. We specifically propose to develop these two applications in parallel to model genetic disease by 1) developing multiple transgenic strains which have transposons in different regions of the genome to take advantage of local hopping for transposon mutgenesis within QTL regions and 2) develop the SB transposon system for somatic mutagenesis in the rat. We chose the Dahl salt-sensitive (SS) inbred rat strain as our model because it is at the forefront of cardiovascular research among many researchers and we have preliminarily characterized it as a new model for human breast cancer. We will combine the SS strain and SB transposon mutagenesis with decades of physiological and pathological characterization to create the disease models and discover the disease genes which are involved. Narrative: The key for researchers to develop diagnostic testing and treatments for complex human diseases like cardiovascular disease and cancer is finding the genes that are responsible. We use what we call `genetic model organisms' like rats to understand the pathology of different diseases and work hard to discover genes that are involved by mutating, or removing them, and determining what happens. We are developing a powerful new tool which is much more efficient at doing this in the rat so that we can accelerate research in many disease areas to improve the quality of life for all of us.