Function follows (iso)form: Exploring functional diversity of isoform expression in single cells

单细胞基因表达分析具有解析构成复杂生物系统的多种细胞类型的能力。大多数基因有可能被选择性地剪接成一种以上的亚型，同一基因的不同亚型编码不同的蛋白质，具有不同的功能。我们假设，这种在大多数单细胞分析中未见的异构体表达的多样性，是单个细胞功能异质性的一种方式。本项目将探索小鼠造血系统中异构体表达的多样性，目的是识别功能异质性异构体及其在单个细胞中的表达模式。使用长阅读测序，我们已经证明了选择性剪接在许多与血细胞发育有关的关键基因中普遍存在。然而，这些异构体在单个细胞中表达的多样性是完全未知的。我们实验室的初步数据表明，单个血液干细胞可以同时表达同一基因的几种异构体，包括那些具有潜在拮抗功能的异构体。我们希望在这个项目中研究这些具有功能意义的选择性剪接事件。利用一系列技术，包括流式细胞仪、单细胞基因组学、下一代短读和长读测序以及生物信息学分析，学生将专注于识别单个血液干细胞和祖细胞中功能相关的异构体表达。然后将通过基因组编辑来研究异构体表达的功能效应，以去除外显子并迫使细胞表达特定的异构体集合，然后通过基因表达、体外和体内分析来监测细胞的反应。

Single-cell gene expression analysis has the power to resolve the multitude of cell types that comprise a complex biological system. The majority of genes have the potential to be alternatively spliced into more than one isoform, with different isoforms of the same gene encoding distinct proteins with distinct functions. We hypothesise that this diversity in isoform expression, unseen in most single-cell analyses, is one way that individual cells can become functionally heterogeneous.This project will explore the diversity of isoform expression in the mouse haematopoietic system, with the aim of identifying functionally heterogeneous isoforms and their pattern of expression within individual cells. Using long-read sequencing, we have demonstrated that alternative splicing is prevalent in many of the key genes involved in blood cell development. However, the diversity of expression of these isoforms is completely unexplored in individual cells. Preliminary data from our lab suggests that single blood stem cells can express several isoforms of a gene simultaneously, including those with potentially antagonistic functions. It is these functionally meaningful alternative splicing events we wish to study in this project.Using a range of techniques, including FACS, single-cell genomics, next generation short- and long- read seqeuncing and bioinformatic anaysis, the student will focus on the identification of functionally consequential isoform expression within individual blood stem and progenitor cells. The functional effect of the isoform expression will then be studied through genome editing to remove exons and force cells to express specific sets of isoforms, then monitoring the cell's response through gene expression, in vitro and in vivo assays.