A novel Drosophila platform for sequential genetic manipulations in vivo

项目总结 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。这项提议充分利用了这一优势，并推动了 果蝇遗传学建立一个新颖、创新和雄心勃勃的平台，允许顺序引入 基因操作--每个操作都与一种不同的荧光蛋白结合--进入单个细胞。我的实验室是 有兴趣利用这个平台建立和研究由成体果蝇肠道组成的肿瘤 具有不同突变特征的遗传异质性细胞群体(即亚克隆)。我们之前已经 以肿瘤序列数据为蓝图，通过基因操作构建多个结直肠癌模型 果蝇在成年苍蝇肠道中人类癌症驱动基因的同源基因。我们现在寻求扩大这些努力 建立新的癌症模型，以反映通过测序和 计算研究。 肿瘤是由癌症驱动基因中基因组变化的顺序积累而产生的。因此，大多数 实体肿瘤是由具有不同突变特征的亚克隆组成的，它们不断地争夺有限的空间。 和资源。治疗往往通过引入额外的选择压力来改变亚克隆的动态，最终 导致抗性亚克隆的扩张和治疗失败。建立亚克隆性肿瘤是一项技术上的 具有挑战性的问题，需要复杂的基因操作，代表着 未得到满足的医疗需求。果蝇是一种有用的癌症模型，它捕捉到了人类肿瘤的几个关键特征， 包括结直肠癌。成年果蝇的肠道是一种具有多潜能的组织。 在结肠肿瘤中信号通路改变的干细胞非常保守，特征也很好。 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 以产生在人类肿瘤中观察到的具有不同亚克隆结构的肿瘤。如果成功，这项技术 将通过提供一个从功能上探索的实验平台来补充哺乳动物癌症模型 癌症大数据揭示了人类肿瘤基因组图景的另一层复杂性。我们也希望 使用这个平台来探索改变癌症驱动基因组改变的顺序对 肿瘤的发生和不同类型的肠道细胞作为起源的肿瘤细胞的潜力。 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，以 产生携带独特荧光蛋白条形码的不同遗传操作的细胞群 在任何感兴趣的组织中的结合。如果成功，它可能会在其他研究中打开有希望的机会 包括发育生物学、干细胞生物学和神经科学的领域，通过提供一个平台来探索 在正常组织功能和疾病背景下的细胞竞争和合作机制。

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.