EMBRYONIC NEURONAL CDNA LIBRARIES: TRANSGENIC ZEBRAFISH

胚胎神经元 CDNA 文库：转基因斑马鱼

• 批准号: 6387756
• 负责人: Shuo Lin
• 金额: $ 3.29万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387756
• 关键词: RNA directed DNA polymerase; animal genetic material tag; cell sorting; complementary DNA; embryo /fetus cell /tissue; gene expression; gene mutation; genetic library; genetically modified animals; genotype; green fluorescent proteins; in situ hybridization; molecular biology information system; neurogenesis; neurons; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; radiation genetics; sequence tagged sites; zebrafish

It is important that an Expressed Sequence Tag (EST) database produced for a model organism represents rnRNA transcripts from all possible tissues. We propose to use a transgenic approach to help identify cell lineage-specific ESTs from developing zebrafish embryos. The focus of this proposal will be the identification of ESTs from embryonic neuronal cells. To this end, we have generated transgenic zebrafish that express the green fluorescent protein (GFP) reporter gene specifically in embryonic neuronal cells. GFP-positive neuronal cells will be purified from living embryos using fluorescence activated cell sorting (FACS). RNA isolated from the FACS-purified cells will then be used to construct cDNA libraries. These libraries will be submitted to the NIH-sponsored Washington University EST Center for large scale DNA sequencing analysis and sequence information will be entered into public database directly from the EST center. We will search the database with our EST sequences to "weed out" those genes already identified. EST sequences whose function is not yet identified will be considered to be "novel" and will be used to analyze expression patterns and chromosomal locations. Genes identified in these experiments, coupled with the knowledge generated concerning their expression patterns and chromosomal assignment, should serve as excellent candidate genes for the zebrafish embryonic mutations affecting neuronal development.

重要的是，为模型生物生成的表达序列标签 (EST) 数据库代表来自所有可能组织的 mRNA 转录本。我们建议使用转基因方法来帮助识别发育中的斑马鱼胚胎的细胞谱系特异性 EST。 该提案的重点是从胚胎神经元细胞中鉴定 EST。为此，我们培育了在胚胎神经元细胞中特异性表达绿色荧光蛋白（GFP）报告基因的转基因斑马鱼。使用荧光激活细胞分选 (FACS) 从活胚胎中纯化 GFP 阳性神经元细胞。从 FACS 纯化的细胞中分离的 RNA 随后将用于构建 cDNA 文库。这些文库将提交给 NIH 资助的华盛顿大学 EST 中心进行大规模 DNA 测序分析，序列信息将直接从 EST 中心输入公共数据库。我们将使用我们的 EST 序列搜索数据库，以“淘汰”那些已识别的基因。功能尚未确定的 EST 序列将被认为是“新颖的”，并将用于分析表达模式和染色体位置。这些实验中鉴定的基因，加上有关其表达模式和染色体分配的知识，应该成为影响神经元发育的斑马鱼胚胎突变的优秀候选基因。