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: 9231562
• 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/9231562
• 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; Mining; Modeling; Names; Natural regeneration; Newts; Organism; Preparation; Process; 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; empowered; gene discovery; genetic signature; genome editing; genome sequencing; in vivo; insight; limb regeneration; novel; post-doctoral training; promoter; quantum; regenerative; restoration; skills; tool; transcriptome; transcriptomics; whole genome

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.

这个为期三年的博士后培训计划的目标是赋予申请人 在美国建立一个研究再生的独立实验室所需的技能和网络 蝾螈（蝾螈）。蝾螈是再生研究景观的关键组成部分，因为它们 失去后更换复杂身体部位的能力。肢体再生是这方面的一个重要模型。中央 蝾螈肢体再生是一个芽基的形成;一个异质的细胞群，发展成 新的肢体尽管它是中心，但我们对芽基的了解很少， 表达标记。申请人将鉴定调控芽基的基因和顺式调控元件 伊比利亚蝾螈中的芽基形成，并开发荧光表达标记物来鉴定 vivo.这将通过以下研究计划来实现。首先，功能相关的基因组区域 将通过全基因组测序和选择性测序相结合的方式进行测序和组装 开放染色质（Aim 1）。其次，使用单细胞转录组学，细胞类型有助于芽基在 将被分类并识别标记基因。此外，通过整合这种单细胞转录组学， 与获得的基因组序列，顺式调控元件，是过度或不足的代表， 将鉴定芽基标记基因（目的2）。第三，利用CRISPR/Cas9基因组编辑， 将开发表达荧光芽基标记的蝾螈（目标3）。所鉴定的基因，顺式调节基因， 元素和转基因蝾螈将提供前所未有的知识和工具，进一步了解 再生监管，从而使申请人能够开始有前途的独立职业生涯 再生研究