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

描述（由申请人提供）：基于转座子的插入诱变筛选已成功用于许多研究，以诱导果蝇突变。最近，转座子和逆转录病毒为基础的插入突变筛选已被用于在小鼠中，以确定新的候选肿瘤抑制基因和癌基因中存在的体细胞肿瘤。这种方法在今天尤其重要，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。尽管它的承诺，在小鼠中可用的遗传工具的限制，以及与哺乳动物实验相关的费用提出了重大障碍，大规模应用这种方法。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相结合，开发一种用于大规模F1体细胞筛选的通用方法，以识别新的癌基因和肿瘤抑制因子。具体来说，我建议使用条件修饰的piggyBac转座子创建插入突变，这将导致基因失活或异位表达在特定组织（眼睛和肠道）的F1代动物。通过使用Gal 4-UAS系统表达piggyBac转座酶来实现piggyBac元件在特定组织中的动员。依赖于它们的整合位点，piggybac元件在某些情况下会使一些基因重叠。此外，因为我将使用含有UAS序列的piggyBac元件，在某些情况下，插入的元件将导致Gal 4存在下附近基因的表达。此外，为了增加事件的频率，将使用携带多个携带UAS的piggyBac转座元件的弹药染色体。最后，为了允许检测隐性肿瘤抑制基因，FLP-FRT染色体将用于产生由piggyBac元件靶向的染色体的纯合克隆。这些筛选将在两个已建立的模型中进行，以研究肿瘤表型，即眼睛和成人肠道，以比较导致不同组织中肿瘤表型的突变谱。将筛选动物是否存在肿瘤，使用下一代测序（NGS）分析肿瘤，以鉴定受影响的基因。最后，验证这些实验中鉴定的基因将确定它们在哪些途径中起作用。总之，这些研究不仅将确定肿瘤抑制因子和癌基因，而且还提供了关于不同组织中肿瘤表型之间差异的重要信息。

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.