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

项目摘要 功能性癌症基因组学领域中的一个重大挑战是建立实验系统， 生物相关性，经济性和规模，以深入了解不同遗传事件的相互作用， 癌症的发生和发展。我们开发了下一代功能性癌症基因组学检测， 结合了来自条形码慢病毒文库的随机、复合遗传扰动的有效递送 编码功能获得和/或功能丧失事件; 单细胞DNA扩增子测序分析，以计数用于大规模平行测定的慢病毒条形码。 基因型与表型的关联（组织学）。在这里，我们将大大改善这个原型 通过修饰慢病毒构建体，使得它们掺入并转录DNA条形码， 通过单细胞RNA测序捕获，从而能够同时单细胞测定 慢病毒条形码（基因型）和转录组（表型）。为了实现这一目标，我们将设计DNA 条形码和猿猴病毒40多聚腺苷酸化信号序列插入绿色 荧光蛋白（GFP），其由内部巨细胞病毒（CMV）启动子表达。我们亦会加强 通过将普遍存在的染色质开口放置在细胞中， CMV启动子上游的UCOE元件。此外，我们还将确定与以下方面相关的技术参数： 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 条形码并基于来自确定的细胞混合物的转录谱区分细胞群体。一旦 优化后，这项技术将被纳入建立小鼠前列腺上皮转化 试验证明这种方法的原理证明，并定义致癌事件如何协同作用， 赋予不同的前列腺癌细胞状态。这种多样性补充将支持Gerardo Javier， 华盛顿大学的一名学生，他在约翰·K. 李，医学博士，弗雷德哈奇博士。他还将获得共同导师从麦加里霍顿，医学博士谁是高级 弗雷德·哈奇的教师已经制定了一项培训计划，以确保Gerardo的充实与以下方面有关： 科学生产力、教育、沟通技能、专业发展、网络和推广 在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 一名研究生专注于癌症生物学，他最终过渡到博士后研究金，因为他 继续他的职业生涯轨迹，成为一名有影响力的独立癌症研究人员。

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.