Functional genomic analysis of neural crest development

神经嵴发育的功能基因组分析

• 批准号: 6227981
• 负责人: William J Pavan
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6227981
• 关键词: cell line; complementary DNA; developmental genetics; developmental neurobiology; gene expression; gene mutation; genetic techniques; in situ hybridization; messenger RNA; molecular cloning; neural crest; neurogenesis; neurogenetics; tissue /cell culture; transfection

To identify potential SOX10 target genes, we will analyze the effects of altered SOX10 expression on the expression of multiple genes simultaneously using cDNA expression microarrays. Successful microarray experiments require: 1) appropriate mRNA populations to compare, 2) appropriate cDNA populations on the array, 3) critical secondary screens, and 4) defined functional assays. 1) We will compare expression patterns from cell cultures with altered SOX10 function. These will include primary cultures of SOX10Dom/+ versus +/+ neural crest and NC-Ms as well as cell lines transduced with SOX10 expressing viruses. 2)We are developing a standard set of mouse cDNA clones to use in microarray expression analyses based on the WASHU-Merck mouse ESTs. However, we have found that these generalized arrays may not be the most efficient source of cDNA for a particular tissue source. Therefore, we developed a novel strategy to isolate a set of cDNA clones representing the majority of genes that regulate NC-M development and function (Loftus et al, 1999). We will use this set for expression profiling in collaboration with Dr. Trent and the NHGRI microarray core. 3) Since we are screening for target genes of SOX10 transcriptional regulation, we will assess candidates by in situ hybridization using mutant SOX10 embryos at the time when SOX10 expressing cells are present but failing to migrate. Genes exhibiting a NC expression pattern in WT but not in mutant SOX10 expressing cells will be analyzed further. 4) Most in vivo approaches for defining gene function (transgenic and knockout strategies) require extensive cloning, viable offspring, and animal breeding. We will use our NC infection strategy described above (RCAS) to misexpress candidate gene products in NC and examine the effects on development of specific lineages. We have already identified one gene that results in a 4-fold increase in melanocyte number when melanoblasts are specifically targeted. - biotechnology research, cancer research, digestive diseases, gene mapping(non-human), neuroscience, pediatric research

为了鉴定潜在的SOX10靶基因，我们将利用cDNA表达芯片分析SOX10表达改变对多个基因同时表达的影响。成功的微阵列实验需要：1)合适的mRNA群体进行比较，2)合适的cDNA群体在阵列上，3)关键的二次筛选，4)明确的功能分析。1)我们将比较SOX10功能改变的细胞培养的表达模式。这些将包括SOX10Dom/+与+/+神经嵴和NC-Ms的原代培养，以及用表达SOX10的病毒转导的细胞系。2)我们正在开发一套标准的小鼠cDNA克隆，用于基于WASHU-Merck小鼠ESTs的微阵列表达分析。然而，我们发现这些通用阵列可能不是特定组织来源的最有效的cDNA来源。因此，我们开发了一种新的策略来分离一组cDNA克隆，这些克隆代表了调节NC-M发育和功能的大多数基因（Loftus et al, 1999）。我们将与Trent博士和NHGRI微阵列核心合作，使用该集合进行表达谱分析。3)由于我们正在筛选SOX10转录调控的靶基因，我们将在SOX10表达细胞存在但无法迁移的情况下，利用突变的SOX10胚胎进行原位杂交，评估候选基因。在WT中显示NC表达模式而在SOX10突变表达细胞中不显示NC表达模式的基因将进一步分析。4)大多数确定基因功能的体内方法（转基因和敲除策略）需要广泛的克隆，可存活的后代和动物育种。我们将使用上述NC感染策略（RCAS）在NC中错误表达候选基因产物，并检查对特定谱系发展的影响。我们已经确定了一种基因，当黑色素母细胞被特异性靶向时，它会导致黑素细胞数量增加4倍。-生物技术研究、癌症研究、消化系统疾病、基因定位（非人类）、神经科学、儿科研究