Gene Discovery and Expression Analysis using SAGE

使用 SAGE 进行基因发现和表达分析

• 批准号: 6952966
• 负责人: Denise Garcia
• 金额: $ 14.8万
• 依托单位: CALIFORNIA STATE UNIVERSITY SAN MARCOS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6952966
• 关键词: Malacostraca; Parvoviridae; alternatives to animals in research; gene expression; gene expression profiling; genetic library; immunogenetics; molecular cloning; nucleic acid sequence; serial analysis of gene expression; virus cytopathogenic effect

DESCRIPTION (provided by applicant): The primary goal of this project will be to monitor global gene expression patterns in response to Infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in Penaeus stylirostris. Changes in gene expression patterns have profound effects on biological functions. Natural processes within living organisms are coordinated by variations in many genes and their products (RNA and proteins) and are at the core of altered pathologic responses. Increasingly, focused researched efforts are emerging to understand invertebrate defense systems and host response to pathogens. The innate immune strategies developed within invertebrate systems serve as models for understanding host defense strategies in response to viral pathogens for both vertebrate and invertebrates. Currently, there are only two published reports in penaeids using expression analysis to discover genes involved in the immune system. These studies demonstrated increased regulation of previously identified defense peptides using the expressed sequence tag (EST) method. While useful, ESTs are more limited in their scope in that they do not generate comprehensive gene expression profiles. There are two techniques available for global gene expression analysis, cDNA microarray and serial analysis of gene expression (SAGE). Both are aimed at monitoring thousands of genes within a biological system under different experimental conditions, however, SAGE is the only technique that does not require prior knowledge of the genome of interest. In addition, SAGE analysis is based on the direct numerical counting of gene tags for comparison between libraries, rather than the arduous statistical methods required for microarray fluorophore signal intensity analysis. With this in mind, the objectives of this research are to compare global gene expression profiles between virally infected and healthy shrimp using SAGE. This will involve the creation of two SAGE libraries and sequencing approximately 400 clones, representing 16,000 different gene tags, from each library. The fulfillment of these objectives represents a unique opportunity to understand the invertebrate immune system as it relates to viral pathogenesis and will serve as a model to understand other animal, vertebrate and invertebrate, defense mechanisms.

描述（由申请人提供）：本项目的主要目标是监测感染性皮下和造血组织坏死病毒（IHHNV）感染后长吻对虾的整体基因表达模式。基因表达模式的变化对生物功能有着深远的影响。生物体内的自然过程由许多基因及其产物（RNA和蛋白质）的变异协调，是改变病理反应的核心。越来越多的重点研究工作正在出现，以了解无脊椎动物的防御系统和宿主对病原体的反应。在无脊椎动物系统内开发的先天免疫策略作为理解脊椎动物和无脊椎动物响应病毒病原体的宿主防御策略的模型。目前，只有两个发表的报告，在对虾使用表达分析，以发现参与免疫系统的基因。这些研究表明，使用表达序列标签（EST）方法增加了先前鉴定的防御肽的调节。虽然有用，但EST在其范围上更受限制，因为它们不产生全面的基因表达谱。基因表达谱分析主要有两种技术：cDNA微阵列和基因表达系列分析（SAGE）。这两种方法都是为了在不同的实验条件下监测生物系统中的数千个基因，然而，SAGE是唯一一种不需要事先了解感兴趣的基因组的技术。此外，SAGE分析是基于基因标签的直接数字计数，用于文库之间的比较，而不是微阵列荧光团信号强度分析所需的艰苦的统计方法。考虑到这一点，本研究的目标是使用SAGE比较病毒感染虾和健康虾之间的全球基因表达谱。这将涉及创建两个SAGE文库，并对每个文库中代表16，000个不同基因标签的约400个克隆进行测序。这些目标的实现代表了了解无脊椎动物免疫系统的独特机会，因为它与病毒发病机制有关，并将作为了解其他动物，脊椎动物和无脊椎动物防御机制的模型。