Insertional Mutagenesis Strategies in Xenopus tropicalis

热带爪蟾的插入诱变策略

• 批准号: 6733566
• 负责人: PAUL E MEAD
• 金额: $ 40.65万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6733566
• 关键词: Retroviridae; Xenopus; Xenopus oocyte; biotechnology; developmental genetics; diploidy; gene expression; genetic library; genetic mapping; genetic models; genetic screening; genetically modified animals; genome; green fluorescent proteins; hematopoiesis; hematopoietic stem cells; method development; microinjections; model design /development; molecular cloning; mutant; nonmammalian vertebrate embryology; reporter genes; site directed mutagenesis; transfection /expression vector; transposon /insertion element

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.

了解早期脊椎动物发育的分子机制仍然是生物科学的中心目标。 两栖动物作为脊椎动物发育模型系统的使用可以追溯到100多年前。 在过去的50年里，最广泛使用的两栖动物是非洲爪蟾。 X.的生化和发育研究。leevis改变了我们对脊椎动物早期胚胎发育的看法。 尽管该模型系统的实用性，遗传研究是不可行的，在这个物种由于长世代时间（约1年）和四倍体基因组。 最近，X.已提出将非洲乳鼠作为遗传分析的替代模型动物。 X. tropicalis是二倍体，世代时间短（大约4个月），此外，还具有X的特征。laevis是一个强大的发展模式。 我的实验室将在X中使用插入突变方法。tropicalis来鉴定在早期发育中起关键作用的基因。 虽然不如化学诱变策略有效，但插入方法具有在突变位点引入稳定的遗传标签的重要优势。 该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，将可遗传的遗传变化引入X。tropicalis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。我的实验室将关注的生物学问题是造血系统的早期发育。 脊椎动物造血干细胞（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工具。将克隆和鉴定影响发育中胚胎血液产生的突变体和基因陷阱。 这项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。