Insertional Mutagenesis in Zebrafish by SB Transposons

描述：（申请人的摘要）本项目的目标是开发和 使用睡美人（SB）转座子系统来递送各种 基因陷阱、增强子陷阱和聚腺苷酸陷阱构建体，用于递送 插入突变和基因标签。斑马是一种几乎 发育遗传学理想脊椎动物模型系统。然而，在这方面， 缺乏基因插入失活的复杂机制， 斑马鱼这些方法可以允许（1）快速分离与基因表达相关的基因。 与表型异常，（2）鉴定与表型异常相关的基因， 特定组织和器官的正常生长和发育（无论是 表型反应是否明显）和（3）动物品系的发育 这些标记基因对其他基因突变的反应可以 检测和评估。转座子介导的插入失活 染色体基因是低等生物中重要的遗传工具;然而， 直到我们发明了睡美人（SB）转座子系统， 高活性DNA转座子。在本申请中，我们 提出扩展SB转座子系统用于插入诱变， 基因标签以及创建斑马鱼，可用作传感器， 其他基因的突变对正常发育很重要。我们将使用各种 含有荧光蛋白基因的转座子， 这将指导荧光蛋白的表达， 转座子插入到基因中。因此，调查人员将 能够预先确定基因的组织和时间特异性， 被我们的转座子标记，这将允许更多的定向基因 卡位由于其在早期开发过程中始终具有光学透明度， 在转座子捕获载体中使用荧光标记物特别 适用于斑马鱼。 我们建议实现以下具体目标，以实现 上述目标：1）评估捕获基因的效率， 转座子诱捕载体2)开发SB转座子陷阱， 用于检测表达低水平转录物的基因的灵敏度。第三章 建立表达SB转座酶的斑马鱼品系以提高效率 转座子标记的方法4)测量本地换位的效率， 已经含有转座子陷阱载体的斑马线。5)发展 斑马鱼品系，其基因含有转座子陷阱， 斑马鱼的遗传路径图谱。

DESCRIPTION: (Applicant's Abstract) The goal of this project is to develop and use the Sleeping Beauty (SB) Transposon System for delivery of a variety of gene-trap, enhancer-trap, and poly(A)-trap constructs for delivery of insertional mutagenesis and gene-tagging in zebrafish. Zebralish are a nearly ideal vertebrate model system for developmental genetics. However, sophisticated mechanisms for insertional inactivation of genes are lacking in zebrafish. Such methods can permit (1) the rapid isolation of genes associated with phenotypic abnormalities, (2) identification of genes involved with the normal growth and development of specific tissues and organs(whether a phenotypic response is evident or not) and (3) development of lines of animals that have marked genes whose responses to mutations in other genes can be detected and evaluated. Transposon-mediated insertional inactivation of chromosomal genes is a promenent genetic tool in lower organisms; however, until we developed the Sleeping Beauty (SB) Transposon System, there were no highly active DNA transposons available for fish. In this application ,we proposed to extend the SB transposon system for insertional mutagenesis and gene tagging as well as creation of zebrafish that can be used as sensors for mutations in other genes important to proper development. We will use a variety of transposons that contain flourescent protein genes with regulatory sequences that will direct the expression of the flourescent proteins whenever the transposons insert themselves into genes. As a result, investigators will be able to pre-determine the tissue and temporal specificity of a gene that has been tagged by one of our transposons, which will permit more directed gene screens. Owing to its optical clarity at all times during early development, the use of flourescent markers in transposon-trap vectors is particularly suitable for use in zebrafish. We propose to accomplish the following specific aims in order to achieve the above goals: 1) Evaluate the efficiencies of trapping' genes with transposon-trap vecotrs. 2) Develop SB transposon traps with greater sensitivities for detection of genes expressing low levels of transcripts. 3) Establish lines of zebrafish that express SB transposase to improve efficiency of transposon tagging. 4) Measure the efficiencies of local transposition in lines of zebratish that already contain transposon-trap vectors. 5) Develop lines of zebrafish that have genes containing the transposon-traps for further mapping of genetic pathways in zebrafish.