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Characterization of neural crest gene expression using a new transgenic Xenopus m

使用新的转基因非洲爪蟾来表征神经嵴基因表达

基本信息

批准号：
8526449
负责人：
Shuo Wei
金额：
$ 10.66万
依托单位：
WEST VIRGINIA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-08 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8526449
关键词：
Achievement Adoption Affect Amphibia Animal Model Animals Candidate Disease Gene Cells Cephalic Cleft Lip Cleft Palate Complex Congenital Abnormality Data Defect Detection Development Developmental Process Diagnostic Diploidy Disease Dissection Effectiveness Embryo Embryonic Development Ephrins Epithelium Ethylnitrosourea Face Fluorescence Fluorescence-Activated Cell Sorting Future Gene Expression Gene Expression Profile Genes Genetic Screening Goals Head Human Image Knowledge Label Lead Life Maintenance Maps Methodology Methods Molecular Molecular Profiling Monitor Morphogenesis Mutagenesis Mutation Nature Neural Crest Neural Crest Cell Neural tube Newborn Infant Outcome Pathway interactions Pattern Population Proteins Publishing Rana Regulation Reporter Reporting Research Research Project Grants Resolution Role Site Staging Structure Study models Syndrome Tadpoles Techniques Testing Therapeutic Time Tissues Transgenes Transgenic Organisms Vertebrates Work Xenopus base blastomere structure cDNA Arrays cleft lip and palate craniofacial expectation fluorescence imaging gain of function in utero in vivo interest migration prevent programs promoter research study skull base therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): Craniofacial morphogenesis is a complicated developmental process that is controlled by many genes. In vertebrates, the majority of craniofacial structures are derived from the cranial neural crest (CNC), and abnormalities in CNC development often lead to craniofacial disorders. Such disorders are common in humans, with an occurrence rate of one per 700 newborns and hundreds of different syndromes reported to date. The long-term objective of this proposed research is to understand the molecular bases of CNC development and craniofacial disorders. Toward this objective a transgenic Xenopus tropicalis line that expresses fluorescent reporter in the neural crest lineage (the snail2-egfp line) has been generated for live imaging of CNC development. The goal of the current application is to test the application of this transgenic line in detecting CNC defects and purifyig CNC cells for molecular studies. The application has two specific aims. In the first aim, the snail2-egfp line will be further characterized and assessed for its usage in real-time detection of defects in early and late CNC development. The integration site of the transgene insertion will be mapped, and experiments will be carried out to determine if the transgene has any effect on CNC development or expression of nearby genes. To test if the transgenic line is suitable for real-time detection of aberrant CNC development, deficiencies in the CNC will be induced at different stages and monitored by using high- resolution fluorescence imaging. In the second aim, the snail2-egfp line will be used to isolate high-purity CNC cells for transcriptome analyses. Several methods, including fluorescence activated cell sorting, will be evaluated for their effectiveness in purifying EGFP-labeled CNC cells. Purified CNC cells collected at different developmental stages will be used for cDNA microarray studies to identify genes that are dynamically regulated in premigratory CNC cells. Genes of interest will be selected and their expression patterns will be validated by other molecular techniques; some of these genes will be further tested for their potential functions in CNC development in future studies. Thus the expected outcomes of the proposed research project include identification of new genes that are potentially important for CNC development, as well as establishment of a new methodology for direct detection of CNC defects in live embryos. The successful achievement of these outcomes will provide us new tools and knowledge in craniofacial research.

描述（申请人提供）：颅面形态发生是一个复杂的发育过程，受许多基因控制。在脊椎动物中，大多数颅面结构源自颅神经嵴（CNC），CNC发育异常常常导致颅面疾病。此类疾病在人类中很常见，每 700 名新生儿中就有 1 人患有此类疾病，迄今为止已报告了数百种不同的综合症。这项研究的长期目标是了解 CNC 发育和颅面疾病的分子基础。为了实现这一目标，已经生成了在神经嵴谱系中表达荧光报告基因的转基因热带爪蟾品系（snail2-egfp品系），用于 CNC 发育的实时成像。当前应用的目标是测试该转基因系在检测 CNC 缺陷和纯化 CNC 细胞以进行分子研究方面的应用。该应用程序有两个具体目标。在第一个目标中，将进一步表征和评估 snail2-egfp 系列在实时检测中的用途 CNC 开发早期和后期的缺陷。将绘制转基因插入的整合位点，并进行实验以确定转基因是否对 CNC 发育或附近基因的表达有任何影响。为了测试转基因系是否适合实时检测异常的 CNC 发育，将在不同阶段诱导 CNC 的缺陷，并使用高分辨率荧光成像进行监测。在第二个目标中，snail2-egfp 系将用于分离高纯度 CNC 细胞以进行转录组分析。将评估包括荧光激活细胞分选在内的多种方法在纯化 EGFP 标记的 CNC 细胞方面的有效性。在不同发育阶段收集的纯化 CNC 细胞将用于 cDNA 微阵列研究，以识别在迁移前 CNC 细胞中动态调节的基因。将选择感兴趣的基因，并通过其他分子技术验证其表达模式；在未来的研究中，将进一步测试其中一些基因在 CNC 开发中的潜在功能。因此，拟议研究项目的预期成果包括鉴定对 CNC 发育潜在重要的新基因，以及建立直接检测活胚胎 CNC 缺陷的新方法。这些成果的成功实现将为我们提供颅面研究的新工具和知识。