Insertional Mutagenesis Strategies in Xenopus tropicalis

了解脊椎动物早期发育的分子机制仍然是生物科学的中心目标。使用两栖动物作为脊椎动物发育模型系统的历史可以追溯到100多年前。在过去的50年里，使用最广泛的两栖动物是非洲爪蛙，非洲爪蛙。莱维氏X.laevis的生化和发育研究改变了我们看待脊椎动物早期胚胎发育的方式。尽管这种模式系统很有用，但由于世代时间较长(约一年)和四倍体基因组，在该物种中进行遗传研究是不可行的。最近，莱维氏X.laevis的一个近亲被提议作为遗传分析的替代模式动物。热带X.laevis是二倍体，世代时间短(约4个月)，此外，还具有使X.laevis成为强大发育模式的特征。我的实验室将使用插入突变方法在热带疟原虫中识别在早期发育中发挥关键作用的基因。虽然不像化学诱变策略那样有效，但插入方法有一个重要的优势，即在突变部位引入一个稳定的遗传标签。这一特征允许相对简单地克隆被破坏的基因，并且克隆技术不依赖于遗传图谱。我们将使用转座酶和逆转录病毒介导的整合系统将可遗传的基因变化引入热带疟原虫基因组。我们的初步数据表明，这些方法在青蛙身上效果很好。除了诱变筛选，我们还将使用转座子系统来产生基因和增强子陷阱。我的实验室将重点研究的生物学问题是造血系统的早期发育。脊椎动物中的造血干细胞(HSCs)的起源知之甚少。两栖动物遗传模型的建立将为研究HSC形成的早期事件提供重要的工具。影响发育中胚胎血液生产的突变体和基因陷阱将被克隆和鉴定。这项拟议的研究将为正常和疾病状态下的造血生物学提供有价值的见解。

Understanding the molecular mechanisms of early vertebrate development remains a central goal in the biological sciences. The use of amphibians as vertebrate developmental model systems dates back over 100 years. The most widely used amphibian in the last 50 years has been the African clawed frog, Xenopus laevis. Biochemical and developmental studies in X. laevis have changed the way we view the early embryonic development of vertebrates. Despite the utility of this model system, genetic studies are not feasible in this species due to a long generation time (approximately 1 year) and tetraploid genome. Recently, a close relative of X. laevis has been proposed as an alternate model animal for genetic analysis. X. tropicalis is diploid, has a short generation time (approximately 4 months) and, in addition, shares the qualities that has made X. laevis a powerful developmental model. My laboratory will use insertional mutagenesis approaches in X. tropicalis to identify genes that play a critical role in early development. While not as efficient as chemical mutagenesis strategies, insertional methods have the important advantage of introducing a stable genetic tag at the site of the mutation. This feature allows comparatively simple cloning of the disrupted gene and the cloning techniques do not rely on genetic maps. We will use transposase- and retroviral-mediated integration systems to introduce heritable genetic changes into the X. tropicalis genome. Our preliminary data demonstrate that these methods work well in the frog. In addition to mutagenesis screens, we will use the transposon system to generate gene and enhancer traps. The biological question that my laboratory will focus on is early development of the hematopoietic system. Little is known about the origin of hematopoietic stem cells (HSCs) in vertebrates. The development of an amphibian genetic model will provide an important tool to study the early events in HSC formation. Mutants and gene traps that affect the production of blood in the developing embryo will be cloned and characterized. The proposed study will provide valuable insight into the biology of hematopoiesis in both normal and disease states.