Insertional Mutagenesis in Zebrafish by SB Transposons

描述：(申请人摘要)这个项目的目标是开发和 使用睡美人(SB)转座子系统交付各种 基因陷阱、增强子陷阱和聚(A)陷阱的构建 斑马鱼的插入突变和基因标记。斑马是一种近乎 发育遗传学的理想脊椎动物模式系统。然而， 基因插入失活的复杂机制在 斑马鱼。这种方法可以(1)快速分离相关基因 有表型异常的，(2)鉴定与 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 这些标记的基因对其他基因突变的反应可能是 检测和评估。转座子介导的插入失活 染色体基因在低等生物中是一种普遍的遗传工具；然而， 在我们开发出睡美人(SB)转座子系统之前，没有 鱼类可利用的高活性DNA转座子。在此应用程序中，我们 建议扩大SB转座子系统用于插入突变和 基因标记以及创造可用作传感器的斑马鱼 对正常发育很重要的其他基因的突变。我们将使用多种 含有带有调控序列的荧光蛋白基因的转座子 这将指导荧光蛋白的表达 转座子将自己插入到基因中。因此，调查人员将被 能够预先确定具有以下特征的基因的组织和时间特异性 被我们的一个转座子标记，这将允许更多的定向基因 屏幕。由于其在早期发展过程中始终具有光学清晰度， 在转座子陷阱载体中使用荧光标记尤其是 适用于斑马鱼。 我们建议完成以下具体目标，以实现 以上目标：1)评估捕获基因的效率 转座子陷阱向量。2)发展更大的Sb转座子陷阱 检测表达低水平转录本的基因的灵敏度。3) 建立表达SB转座酶的斑马鱼品系以提高效率 转座子标记。4)测量局部转置的效率 已经含有转座子陷阱载体的斑马线虫。5)发展 含有转座子陷阱基因的斑马鱼品系 斑马鱼遗传路径的作图。

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.