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，以寻找对发育、衰老、行为和疾病等特定生物过程重要的基因。虽然通过注射VSV-G伪型小鼠白血病病毒可以在斑马鱼中快速产生数十万种系插入，并且插入突变的致病基因很容易识别，但插入突变尚未在斑马鱼中广泛应用，主要是因为它需要大量的资源来进行大规模的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有效地选择F1中具有有趣表达模式或错误表达表型的基因突变，以进一步分析F2中功能丧失表型。这些诱变剂在整合后不仅会使基因失活，而且还会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，因为病毒更喜欢基因的5'端。初步数据表明，两种类型的载体都能产生高滴度的病毒，这是在斑马鱼中产生病毒诱变原的先决条件。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时确定。该应用的具体目的是：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的系统错误表达筛选；2)开发高滴度、低毒性的基因陷阱病毒，并将其用于基于红色荧光蛋白的表达模式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用于研究界，许多实验室可能会将其用于大规模插入诱变筛选，以识别对他们研究的生物过程重要的基因。从斑马鱼的研究中获得的知识将为我们对人类发展和疾病的理解提供见解。

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.