Large-scale F1 somatic screens for new tumor suppressors and oncogenes in Drosoph

描述（由申请人提供）：基于转座子的插入突变筛选已经在许多研究中成功地用于诱导果蝇突变。最近，转座子和基于逆转录病毒的插入突变筛选已在小鼠中用于鉴定体细胞肿瘤中存在的新的候选肿瘤抑制因子和癌基因。这种方法在今天尤为重要，因为现在很清楚，肿瘤中突变基因的谱是复杂的，并且因组织而异。尽管这种方法很有前景，但小鼠遗传工具的局限性以及哺乳动物实验的费用给这种方法的大规模应用带来了重大障碍。在我的博士后研究期间，我建议将最近哺乳动物筛选的独创性与果蝇中可用的遗传工具结合起来，开发一种用于大规模F1体细胞筛选的通用方法，以识别新的致癌基因和肿瘤抑制因子。具体来说，我建议使用有条件修饰的piggyBac转座子来创建插入突变，从而在F1动物的特定组织（眼睛和肠道）中导致基因失活或异位表达。piggyBac元件在特定组织中的动员将通过使用Gal4-UAS系统表达piggyBac转座酶来完成。在某些情况下，依赖于它们的整合位点，背带元件会使某些基因失活。此外，由于我将使用含有UAS序列的piggyBac元件，在某些情况下，插入的元件将导致Gal4存在时附近基因的表达。此外，为了增加事件发生的频率，将使用携带携带UAS的多个piggyBac转座元件的弹药染色体。最后，为了检测隐性肿瘤抑制基因，将使用FLP-FRT染色体来创建piggyBac元件靶向的染色体的纯合克隆。这些筛选将在两种已建立的模型中进行，以研究肿瘤表型，眼睛和成人肠道，以比较不同组织中导致肿瘤表型的突变谱。研究人员将对动物进行肿瘤筛选，并利用下一代测序技术（NGS）分析受影响的基因。最后，在这些实验中鉴定的基因的验证将确定它们在哪些途径中起作用。总之，这些研究不仅可以识别肿瘤抑制因子和癌基因，还可以为不同组织中肿瘤表型之间的背景差异提供重要信息。

DESCRIPTION (provided by applicant): Transposon-based insertional mutagenesis screens have been used successfully in numerous studies to induce mutations in Drosophila. Recently, transposon and retrovirus-based insertional mutagenesis screens have been used in the mouse to identify new candidate tumor suppressors and oncogenes present in somatic tumors. This approach is especially important today as it is now clear that the spectrum of mutated genes in a tumor is complex and varies from tissues to tissues. Despite its promises, the limitation of the genetic tools available in the mouse together with the expense associated with mammalian experiments present significant obstacles to the large-scale application of this approach. During my postdoctoral studies, I propose to combine the recent ingenuity of the mammalian screens with the genetic tools available in Drosophila to develop a general method for large-scale F1 somatic screens to identify new oncogenes and tumor suppressors. Specifically, I propose to use conditionally modified piggyBac transposons to create insertional mutations that will result in either gene inactivation or ectopic expression in specific tissues (eye and gut) in F1 animals. Mobilization of the piggyBac elements in specific tissues will be accomplished by expressing the piggyBac transposase using the Gal4-UAS system. Depending on their integration sites the piggybac elements in some cases will inactivate some genes. In addition, because I will use piggyBac elements that contain a UAS sequence, in some cases the inserted elements will lead to expression of nearby genes in the presence of Gal4. Further, to increase the frequency of events, an ammunition chromosome that carries multiple piggyBac transposable elements carrying a UAS will be used. Finally, to allow for detection of recessive tumor suppressor genes FLP-FRT chromosomes will be used to create homozygous clones of the chromosomes that are targeted by the piggyBac elements. These screens will be performed in two established models to study tumor phenotypes, the eye and in the adult gut, to compare the spectrum of mutations leading to tumor phenotypes in different tissues. Animals will be screened for the presence of tumors that will be analyzed using Next Generation Sequencing (NGS) to identify the genes affected. Finally, validation of the genes identified in these experiments will identify in which pathways they are acting. Altogether, these studies will not only identify tumor suppressors and oncogenes but also provide important information on the contextual differences between tumor phenotypes in different tissues. PUBLIC HEALTH RELEVANCE: This project is seeking support for the identification of novel tumor suppressors and oncogenes in Drosophila. The result of this study will greatly increase our understanding of which genes, out of the many that are mutated in cancerous tissues, are responsible for causing tumor phenotypes. This work is relevant to human cancer because of the amazing gene conservation between fly and humans.