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Identification of stage-and tissue-specific endogenous tick promoters

阶段和组织特异性内源蜱启动子的鉴定

基本信息

批准号：
10648720
负责人：
Monika Gulia-Nuss
金额：
$ 20.11万
依托单位：
UNIVERSITY OF NEVADA RENO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10648720
关键词：
ATAC-seq Adult Anaplasma Arthropods Babesia Binding Bioinformatics Biological Assay Biology Black-legged Tick Blood Borrelia burgdorferi CMV promoter CRISPR/Cas technology Cell Line Centers for Disease Control and Prevention (U.S.)Chromatin Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats Complex DNA Development Embryo Eukaryotic Cell Eye Color Future Gene Combinations Gene Expression Genes Genomics Goals Heat shock proteins High-Throughput Nucleotide Sequencing Individual Injections Insecta Life Lyme Disease Messenger RNA Methods Modeling Molecular Molecular Conformation Mutation Nuclear Order Spirochaetales Organism Pattern Phenotype Promoter Regions Proteins Protocols documentation Reporter Research Research Personnel Rickettsia Risk Taking Screening procedure Serine Protease System Technology Temperature Testing Ticks Tissues Transcript Transposase United States Validation Vector-transmitted infectious disease Vertebrates Viral Virus Vitellogenins Work differential expression experience feeding functional genomics gene function genetic regulatory protein genome editing genome sequencing genomic locus genomic tools improved in vivo evaluation knockout gene mutant pathogen promoter protocol development screening sex success tool transcriptome transcriptome sequencing transcriptomics transmission process vector

项目摘要

SUMMARY The lack of a promoter-reporter system in ticks makes functional genomics studies challenging. A few exogenous promoters that appear to work in tick cell lines are viral (i.e. CAAG/CMV promoter) and are constitutively expressing. However, not all research questions can be approached by constitutively expressing a gene. It is useful to gain temporal control over gene expression. In eukaryotic cells, nuclear DNA and proteins combine to form chromatin, which then undergoes complex and orderly folding to form chromosomes. For genes to be expressed, chromatin must be in an open conformation. Open chromatin allows regulatory proteins to bind to DNA and regulate DNA function. The assay for transposase-accessible chromatin with high- throughput sequencing (ATAC-seq) enables high-throughput sequencing of open chromatin regions with the help of transposases. ATAC-seq detects chromatin accessibility of related genes and indicates their regulatory mechanisms. Thus, genes with chromatin accessibility in promoters are more likely to be differentially expressed at the mRNA level. Therefore, by combining the power of ATAC-seq to analyze chromatin accessibility in the promoter regions of whole genes in ticks’ tissues and life stages and then screening differentially expressed genes (DEGs) at the mRNA level by transcriptome sequencing technology (RNA-seq), we will obtain temporally and spatially expressed genes and their promoters. Therefore, our hypothesis/ objective is that by combining gene expression (RNAseq) and open chromatin regions using ATACseq (Aim 1), and promoter assays (Aim 2), we will identify constitutive and stage/sex/tissue-specific promoters for gene expression in ticks. Our pioneering work in tick genome sequencing and CRISPR-Cas9-based genome editing has now made tick gene-editing possible and this proposal will further help us improve the gene-editing protocol by developing the promoter-reporter systems that will allow the screening of mutants without the need to sequence every individual.

总结由于蜱类缺乏启动子-报告基因系统，使得功能基因组学研究具有挑战性。一些似乎在蜱细胞系中起作用的外源启动子是病毒启动子（即CAAG/CMV 启动子）并组成型表达。然而，并非所有的研究问题都可以通过组成型表达基因来接近。获得对基因的时间控制是有用的，表情在真核细胞中，核DNA和蛋白质联合收割机形成染色质，然后经过复杂有序的折叠形成染色体。对于要表达的基因，染色质必须处于开放构象。开放的染色质允许调节蛋白结合并调节DNA的功能。高表达的转座酶可接近染色质的测定通量测序（ATAC-seq）实现开放染色质的高通量测序在转座酶的帮助下。ATAC-seq检测相关基因的染色质可及性基因，并指出其调控机制。因此，基因与染色质可及性，启动子更可能在mRNA水平上差异表达。因此通过结合ATAC-seq的力量来分析在启动子区域中的染色质可及性，在蜱的组织和生活史中的全基因，然后筛选差异表达的基因通过转录组测序技术（RNA-seq）在mRNA水平上对DEG进行分析，我们将获得时间和空间表达的基因及其启动子。因此，我们的假设/ 目的是通过结合基因表达（RNAseq）和开放的染色质区域， ATACseq（Aim 1）和启动子测定（Aim 2），我们将鉴定组成型和阶段/性别/组织特异性启动子用于蜱中的基因表达。我们在蜱虫方面的开创性工作基因组测序和基于CRISPR-Cas9的基因组编辑现在已经使蜱虫基因编辑这一提议将进一步帮助我们通过开发基因编辑技术来改进基因编辑方案。启动子-报告子系统，该系统将允许筛选突变体，而不需要给每个人排序