viruses for gene-trap and misexpression screens

描述（由申请人提供）：为响应PAR-02-142“斑马鱼遗传分析工具”，本申请旨在开发两种类型的逆转录病毒诱变剂，使小型实验室能够在斑马鱼中进行大规模插入诱变筛选，以筛选对发育、衰老、行为和疾病中特定生物过程重要的基因。虽然通过注射VSV-G假型小鼠白血病病毒可以在斑马鱼中快速产生数十万个种系插入，并且插入突变体的致病基因很容易识别，但插入诱变尚未在斑马鱼中广泛使用，主要是因为它需要大量资源进行大规模的3代筛选。相比之下，所提出的基因激活和基因陷阱病毒将允许在F1中有效地选择具有感兴趣的表达模式或错误表达表型的基因中的突变，以进一步分析F2中的功能丧失表型。这些诱变剂在整合时不仅会使基因失活，而且还可以用荧光报告基因揭示活鱼中的表达模式，和/或诱导组织特异性错误表达表型，因为病毒偏好基因的5'端。初步数据表明，这两种类型的载体可以产生高滴度病毒，这是斑马鱼中病毒诱变剂的先决条件。这些病毒的效用将在筛选对视网膜发育和功能重要的基因中确定。本申请的具体目的是：1）开发高滴度、低毒性的基因激活病毒，并将其用于视网膜祖细胞中的系统性错误表达筛选; 2）开发高滴度、低毒性的基因捕获病毒，并将其用于基于红色荧光蛋白的表达模式筛选，以筛选对视网膜发育和功能重要的基因。由于病毒诱变剂将可供研究界使用，许多实验室可能会将其应用于大规模插入诱变筛选，以确定对他们研究的生物过程重要的基因。从斑马鱼研究中获得的知识将为我们理解人类发育和疾病提供见解。

DESCRIPTION (provided by applicant): In response to PAR-02-142 "Tools for Genetic Analysis in Zebrafish", this application is to develop two types of retroviral mutagens that would allow small laboratories to perform large-scale insertional mutagenesis screens in zebrafish for genes important for specific biological processes in development, aging, behavior and diseases. Although hundreds of thousands of germline insertions can be generated quickly in zebrafish by injecting VSV-G pseudotyped murine leukemia virus, and causative genes for insertional mutants are readily identifiable, insertional mutagenesis has not been widely used in zebrafish mainly because it requires extraordinary resources for a large-scale, 3-generation screen. In contrast, the proposed gene-activation and gene-trap viruses will allow efficient selection of mutations in genes with interesting expression patterns or misexpression phenotypes in F1 for further analysis of loss-of-function phenotypes in F2. These mutagens will not only inactivate genes when integrated, but also reveal the expression patterns in live fish with a fluorescent reporter, and/or induce tissue-specific misexpression phenotypes, since the virus prefers the 5' end of genes. Preliminary data indicate that both types of vectors can produce high titer viruses, a prerequisite for a viral mutagen in zebrafish. The utilities of the viruses will be determined in screens for genes important for retinal development and function. The specific aims of the application are: 1) develop high titer, low toxicity gene-activation viruses and use them in a systematic misexpression screen in retinal progenitor cells; 2) develop high titer, low toxicity gene-trap viruses and use them in a red fluorescent protein-based expression pattern screen for genes important for retinal development and function. Because the viral mutagens will be available to the research community, many labs may apply them in large-scale insertional mutagenesis screens to identify genes important for the biological processes they study. Knowledge gained from studies in zebrafish will provide insights to our understanding of human development and diseases.