A novel Drosophila platform for sequential genetic manipulations in vivo

项目摘要 进行复杂的基因操作和大规模探索性研究的能力是关键 果蝇作为模型系统的优势。该提案利用了这一优势，并推动了 果蝇遗传学建立一个新颖的，创新的和雄心勃勃的平台，允许顺序引入 基因操作-每一个都与不同的荧光蛋白结合-进入单个细胞。我的实验室是 有兴趣使用这个平台来建立和研究成年果蝇肠道中的肿瘤， 具有不同突变谱的遗传异质性细胞群体（即亚克隆）。我们先前已经 使用肿瘤序列数据作为蓝图，通过基因操作建立几种结直肠癌模型， 成年果蝇肠道中人类癌症驱动基因的果蝇直系同源物。我们现在寻求扩大这些努力 建立新的癌症模型，反映通过测序鉴定的肿瘤亚克隆结构， 计算研究。 肿瘤是通过癌症驱动基因中基因组改变的连续积累而产生的。因此，大多数 实体瘤由具有不同突变谱的亚克隆组成，它们不断竞争有限的空间 和资源治疗通常通过引入额外的选择压力来改变亚克隆动力学， 导致耐药亚克隆的扩增和治疗失败。构建亚克隆肿瘤是一种技术上 具有挑战性的问题，需要复杂的遗传操作，代表了一个重要的领域， 未满足的医疗需求果蝇是一种有用的癌症模型，它捕捉了人类肿瘤的几个关键特征， 包括结肠直肠癌果蝇成虫的肠是一种具有多能性的肠上皮细胞， 在结肠肿瘤中改变了信号通路的干细胞是显著保守的并且被很好地表征。 这里提出的平台，结合果蝇的实际优势，提供了一个独特的机会 以产生具有在人肿瘤中观察到的不同亚克隆结构的肿瘤。如果成功，这项技术 将通过提供一个实验平台来补充哺乳动物癌症模型， 大规模癌症数据揭示了人类肿瘤基因组景观的另一层复杂性。我们也希望 利用这个平台来探索改变癌症驱动基因组改变的顺序对 肿瘤发生和肠的不同细胞类型作为肿瘤起源细胞的潜力。 这里提出的平台是一种灵活的技术，可以很容易地与其他遗传工具相结合， 产生携带用独特荧光蛋白条形码化的不同遗传操作的细胞群 在任何感兴趣的组织中的组合。如果成功，它可以为其他研究开辟有希望的机会 包括发育生物学，干细胞生物学和神经科学领域，通过提供一个平台，探索 在正常组织功能和疾病的背景下，细胞竞争和合作的机制。

PROJECT SUMMARY The ability to perform sophisticated genetic manipulations and large-scale exploratory studies is a key strength of Drosophila as a model system. This proposal leverages this strength and pushes the limits of Drosophila genetics to establish a novel, innovative and ambitious platform that allows sequential introduction of genetic manipulations —each coupled with a different fluorescent protein— into individual cells. My laboratory is interested in using this platform to build and study tumors in the adult Drosophila intestine composed of genetically heterogeneous cell populations with distinct mutation profiles (i.e. subclones). We have previously used tumor sequence data as blueprints to build several colorectal cancer models by genetically manipulating Drosophila orthologs of human cancer driver genes in the adult fly intestine. We now seek to expand these efforts to building new cancer models that reflect tumor subclonal architectures identified by sequencing and computational studies. Tumors arise by sequential accumulation of genomic alterations in cancer driver genes. Consequently, most solid tumors are composed of subclones with distinct mutation profiles in constant competition for limited space and resources. Therapy often alters subclonal dynamics by introducing additional selective pressures, eventually leading to the expansion of resistant subclones and therapy failure. Building subclonal tumors is a technically challenging problem that requires sophisticated genetic manipulations and represents an important area of unmet medical need. Drosophila is a useful cancer model that captures several key hallmarks of human tumors, including colorectal cancer. The adult Drosophila intestine is a well characterized tissue with multipotent intestinal stem cells where signaling pathways altered in colon tumors are remarkably conserved and well characterized. The platform proposed here, combined with practical advantages of Drosophila, provides a unique opportunity to generate tumors with diverse subclonal architectures observed in human tumors. If successful, this technology will complement mammalian cancer models by providing an experimental platform to functionally explore yet another layer of complexity of human tumor genome landscapes revealed by big cancer data. We also hope to use this platform to explore the impact of altering the order of cancer driving genomic alterations on tumorigenesis and the potential of different cell types of the intestine as tumor cells of origin. The platform proposed here is a flexible technology that can easily be combined with other genetic tools to generate populations of cells carrying different genetic manipulations barcoded with unique fluorescent protein combinations in any tissue of interest. If successful, it could open up promising opportunities in other research areas including developmental biology, stem cell biology and neuroscience by providing a platform to explore mechanisms of cell competition and cooperation in the context of normal tissue function as well as disease.