Discovering signature genes and developing expression markers for blastema cells

发现胚基细胞的特征基因并开发表达标记

• 批准号: 9447270
• 负责人: Ahmed Elewa
• 金额: $ 0.89万
• 依托单位: KAROLINSKA INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-25 至 2019-04-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9447270
• 关键词: ATAC-seq; Adult; Ambystoma; Bioinformatics; Body part; Brain Injuries; CRISPR/Cas technology; Cells; Chromatin; Clinical; Complex; Development; Genes; Genome; Genomic Segment; Genomics; Heart; Human; Human body; Knowledge; Laboratories; Libraries; Light; Limb structure; Medicine; Modeling; Names; Natural regeneration; Newts; Organism; Preparation; Process; Promoter Regions; Regulation; Regulatory Element; Reporter; Research; Rodent; Salamander; Stem cells; Surveys; Training Programs; Transcriptional Regulation; Transgenic Animals; Transgenic Organisms; Translational Regulation; Transplanted tissue; United States; Untranslated Regions; Vertebrates; blastema; career; cell type; gene discovery; genetic signature; genome editing; genome sequencing; in vivo; insight; limb regeneration; novel; post-doctoral training; promoter; public health relevance; quantum; regenerative; restoration; skills; tool; transcriptome; transcriptomics; whole genome

 DESCRIPTION (provided by applicant): The objective of this three-year postdoctoral training program is to empower the applicant with the necessary skills and network to establish an independent laboratory in the United States studying regeneration in newts (salamanders). Newts are critical components of the regenerative research landscape due to their ability to replace complex body parts after loss. Limb regeneration is an important model in this regard. Central to newt limb regeneration is the formation of a blastema; a heterogeneous mass of cells that develop into the new limb. Despite its centrality, we have a minimal understanding of the blastema and lack any specific expression markers. The applicant will identify the genes and cis-regulatory elements that regulate blastema formation in the Iberian newt and develop fluorescent expression markers to identify blastema cell types in vivo. This will be accomplished through the following research plan. First, functionally relevant genomic regions will be sequenced and assembled by a combination of whole genome sequencing and selectively sequencing open chromatin (Aim 1). Second, using single-cell transcriptomics, the cell types contributing to the blastema at will be classified and marker genes identified. Additionally, by integrating this single-cell transcriptomic information with obtained genomic sequences, cis-regulatory elements that are over- or under- represented in blastema markers genes will be identified (Aim 2). Third, using CRISPR/Cas9 genome editing, transgenic newts expressing fluorescent blastema markers will be developed (Aim 3). The identified genes, cis-regulatory elements and transgenic newts will offer unprecedented knowledge and tools to further understand the regulation of regeneration, thereby enabling the applicant to begin a promising independent career in regeneration research.

 描述（由申请人提供）：这个为期三年的博士后培训计划的目标是使申请人具备必要的技能和网络，在美国建立一个独立的实验室，研究蝾螈（蝾螈）的再生。蝾螈是再生研究领域的重要组成部分，因为它们有能力在失去后更换复杂的身体部位。肢体再生是这方面的一个重要模型。蝾螈肢体再生的核心是胚基的形成;胚基是发育成新肢体的异质细胞群。尽管它的中心地位，我们有一个最低限度的了解芽，缺乏任何具体的表达标记。申请人将鉴定调控伊比利亚蝾螈芽基形成的基因和顺式调控元件，并开发荧光表达标记物以鉴定体内芽基细胞类型。这将通过以下研究计划来实现。首先，将通过全基因组测序和选择性测序开放染色质的组合对功能相关的基因组区域进行测序和组装（Aim 1）。第二，使用单细胞转录组学，将有助于芽基的细胞类型分类并鉴定标记基因。此外，通过将该单细胞转录组学信息与获得的基因组序列整合，将鉴定在芽基标记基因中过量或不足表达的顺式调节元件（Aim 2）。第三，使用CRISPR/Cas9基因组编辑，将开发表达荧光芽基标记的转基因蝾螈（Aim 3）。鉴定的基因、顺式调控元件和转基因蝾螈将提供前所未有的知识和工具，以进一步了解再生的调控，从而使申请人能够开始一个有前途的再生研究的独立职业生涯。