Diversity Supplement: Uncovering diverse genotype-phenotype relationships in prostate cancer

项目摘要 在功能癌症基因组学领域的一个重大挑战是建立与 生物学相关性、经济性和规模，以获得对不同基因事件相互作用的洞察 癌症的发生和发展。我们开发了一种下一代功能性癌症基因组学分析方法 结合了来自条形码慢病毒文库的随机、复合遗传扰动的高效传递 编码功能获得和/或功能丧失事件；癌症表型的生物选择；以及 单细胞DNA扩增序列分析用于计数大规模平行的慢病毒条形码 基因分型与表型的关系(组织学)。在这里，我们将对此原型进行实质性改进 通过修改慢病毒构建物以使其整合和转录DNA条形码来进行检测 通过单细胞RNA测序捕获，从而实现同时单细胞测定 慢病毒条形码(基因)和转录体(表型)。为了实现这一点，我们将设计DNA 条形码和猴病毒40多聚腺苷信号序列插入绿色基因的3‘非翻译区 由内部巨细胞病毒(CMV)启动子表达的荧光蛋白(GFP)。我们还将加强 通过放置普遍存在的染色质开口来全面转录GFP并缓解CMV启动子的沉默 位于CMV启动子上游的元件(UCOE)。此外，我们将确定与以下相关的技术参数 单细胞RNA测序以确定准确捕获化合物所需的相对读取深度 条形码和基于来自定义的细胞混合物的转录特征的细胞群体的区分。一次 经过优化，这项技术将被整合到已建立的小鼠前列腺上皮转化中 验证这一方法的原理，并定义致癌事件如何协同作用 传递不同的前列腺癌细胞状态。这一多样性补充将支持毕业生杰拉尔多·哈维尔 他是华盛顿大学的一名学生，在约翰·K。 Lee，医学博士，弗雷德·哈奇博士。他还将接受麦克格里·霍顿医学博士的共同指导，霍顿是一名大四学生 弗雷德·哈奇的教员。已经制定了一项训练计划，以确保杰拉尔多的浓缩与 科学生产力、教育、沟通技能、专业发展、网络和晋升 生物医学科学中代表性不足的少数群体。这种支持将有利于他作为一名 一名专注于癌症生物学的研究生，他最终过渡到博士后职位，因为他 继续他成为一名有影响力的独立癌症研究人员的职业轨迹。

Project Summary A significant challenge in the field of functional cancer genomics is to establish experimental systems with biologic relevance, economy, and scale to derive insights into the interactions of diverse genetic events in cancer initiation and progression. We developed a next-generation functional cancer genomics assay that combines efficient delivery of random, compound genetic perturbations from barcoded lentiviral libraries encoding gain-of-function and/or loss-of-function events; biological selection for a cancer phenotype; and single-cell DNA amplicon sequencing analysis to enumerate lentiviral barcodes for the massively parallel association of genotype with phenotype (histology). Here we will substantially improve upon this prototype assay by modifying the lentiviral constructs such that they incorporate and transcribe DNA barcodes that can be captured by single-cell RNA sequencing, thereby enabling simultaneous single-cell determination of lentiviral barcodes (genotype) and transcriptomes (phenotype). To accomplish this, we will engineer DNA barcodes and a simian virus 40 polyadenylation signal sequence into the 3’ untranslated region of a green fluorescent protein (GFP) expressed from an internal cytomegalovirus (CMV) promoter. We will also enhance overall GFP transcription and mitigate CMV promoter silencing by placing a ubiquitous chromatin opening element (UCOE) upstream of the CMV promoter. Further, we will determine technical parameters related to single-cell RNA sequencing to determine the relative read depths necessary to accurately capture compound barcodes and discriminate cell populations based on transcriptional profiles from a defined cell mixture. Once optimized, this technology will be incorporated into an established mouse prostate epithelial transformation assay to demonstrate proof-of-principle of this approach and define how oncogenic events may cooperate to impart distinct prostate cancer cell states. This diversity supplement will support Gerardo Javier, a graduate student at the University of Washington who is conducting research under the primary mentorship of John K. Lee, MD, PhD at Fred Hutch. He will also receive co-mentorship from McGarry Houghton, MD who is a senior faculty member at Fred Hutch. A training plan has been crafted to ensure Gerardo’s enrichment related to scientific productivity, education, communication skills, professional development, networking, and promotion of underrepresented minorities in biomedical sciences. This support will be beneficial to his training as a graduate student focused on cancer biology and his ultimate transition to a post-doctoral fellowship as he continues his career trajectory of becoming an impactful independent cancer researcher.