viruses for gene-trap and misexpression screens

描述(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，使小型实验室能够对斑马鱼进行大规模插入突变筛选，以寻找在发育、衰老、行为和疾病等特定生物学过程中至关重要的基因。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个生殖系插入，并且插入突变的致病基因很容易被识别，但插入突变还没有被广泛应用于斑马鱼，主要是因为它需要大量的资源来进行大规模的3代筛选。相反，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的突变，以便进一步分析F2的功能丧失表型。这些诱变剂不仅在整合时会使基因失活，而且还会通过荧光报告揭示活鱼的表达模式，和/或诱导组织特异性的错误表达表型，因为病毒喜欢基因的5‘端。初步数据表明，这两种载体都能产生高滴度病毒，这是斑马鱼产生病毒诱变剂的先决条件。病毒的效用将在视网膜发育和功能重要基因的筛查中确定。该应用的具体目的是：1)开发高滴度、低毒的基因激活病毒，并将其用于系统的视网膜前体细胞错误表达筛查；2)开发高滴度、低毒的基因陷阱病毒，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。由于病毒诱变剂将提供给研究界，许多实验室可能会将它们应用于大规模插入突变筛选，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.