Sleeping Beauty transposon system for mutagenesis in zebrafish

描述(申请人提供)：自1993年以来，美国的癌症死亡率稳步下降，但总体降幅相对较小，部分原因是对侵袭性和转移性疾病的治疗策略不足。这在一定程度上源于我们对促进癌细胞转移行为的基因变化的不完全理解。缺乏这样的知识阻碍了针对导致转移的改变的细胞路径的新疗法的开发。我们的长期目标是确定驱动肿瘤诱导和转移的遗传途径，以揭示新的治疗靶点。这里的目标是建立一个突变系统，以识别在肿瘤进展到转移过程中起作用的基因。我们之前在斑马鱼中开发了一种强大的转座子体细胞突变策略，该策略可以在成年组织中诱导肿瘤并识别癌症基因。我们建议扩展我们原有的系统，使其既适用于体细胞突变，也适用于生殖系突变。将创建可诱导的和针对生殖系的转座酶来源，以用于现有的Gal4驱动系。我们设计了一种新型的含有荧光报告的基因断裂转座子来捕捉编码和非编码基因的表达。这种荧光报告器将允许在移植到WIL型受体后的肿瘤形成过程中跟踪单细胞。我们将结合我们最近在实验室分离的视网膜肿瘤模型来测试我们的突变系统促进转移行为的能力。转座子整合位点分析将在表现出转移行为的单个细胞上进行。其目标是确定与单个癌细胞转移行为相关的基因途径。我们提出这项工作的基本原理是，我们的突变系统与斑马鱼成人和儿童癌症模型相结合，将识别在肿瘤发生和转移中协同作用的新基因。这一信息有望揭示治疗转移性疾病的新的治疗靶点。

DESCRIPTION (provided by applicant): The U.S. has seen a steady decline in cancer mortality rates since 1993, but the overall decrease is relatively slight, due in part to insufficint treatment strategies for invasive and metastatic disease. This stems in part from our incomplete understanding of the genetic changes that promote metastatic behavior in cancer cells. Lack of such knowledge hinders the development of new therapies to target altered cellular pathways leading to metastasis. Our long-term goal is to define genetic pathways that drive tumor induction and metastasis in order to reveal novel therapeutic targets. The objective here is to build a mutagenesis system that will identify genes that cooperate in tumor progression to metastasis. We previously developed a robust transposon somatic mutagenesis strategy in zebrafish that leads to tumor induction in adult tissues and identification of cancer genes. We propose to expand our original system so it is applicable to both somatic and germ line mutagenesis. Inducible and germ line targeted transposase sources will be created for use with existing Gal4 driver lines. We have designed a novel gene breaking transposon containing a fluorescent reporter to capture expression of coding and noncoding genes. The fluorescence reporter will allow single cells to be followed during tumorigenesis after transplantation into wil type recipients. We will test the ability of our mutagenesis system to promote metastatic behavior in combination with a retinal tumor model we recently isolated in our laboratory. Transposon integration site analysis will be carried out on single cells exhibiting metastatic behavior. The goal is to identify gene pathways that correlate with metastatic behavior of individual cancer cells. Our rationale for the pro- posed work is that our mutagenesis system in combination with zebrafish models of adult and childhood cancers will identify new genes that cooperate in tumor onset and metastasis. This information can be expected to reveal new therapeutic targets for treating metastatic diseases.