Cloning of Zebrafish exocrine pancreas mutant

斑马鱼外分泌胰腺突变体的克隆

• 批准号: 7117651
• 负责人: Nelson Shu-Sang Yee
• 金额: $ 7.2万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7117651
• 关键词: acinar cell; alternatives to animals in research; artificial chromosomes; biological signal transduction; carcinogenesis; cell growth regulation; cell proliferation; developmental genetics; gene expression; genetic mapping; genetic markers; genetic polymorphism; genetic regulation; histogenesis; molecular cloning; mutant; nucleic acid sequence; pancreas; pancreas neoplasms; pigmentation; polymerase chain reaction; site directed mutagenesis; zebrafish

DESCRIPTION (provided by applicant): The long-term objective is to develop strategies for cancer prevention and therapy by understanding the molecular basis of human cancer through studies of developmental and signaling mechanisms in model organisms. Zebrafish pancreas is used as a model organ to identify and characterize developmental regulators of cell fate and lineage determination, proliferation, survival, differentiation, morphogenesis and growth. A strategy has been developed to identify the genes that control various aspects of pancreatic organogenesis through chemical-induced mutagenesis screen that affect pancreas development in zebrafish. One (1)unique mutant, exocrine and pigmentation (eap), has small exocrine pancreas, normal islet and reduced skin pigment. Pancreatic duct branching is hypomorphic, and the acini are relatively small, whereas the acinar cells are differentiated but they contain relatively few zymogen granules. This data has led to our hypothesis that the epd mutation causes growth arrest of the exocrine pancreas. The epd gene product is expected to play a regulatory role in cellular proliferation of exocrine progenitors. The goal of this proposed study is to identify the epd gene by positional cloning technique. The first specific aim is to determine the chromosomal location of the epd gene based on simple sequence length polymorphism, and define the critical region harboring the epd gene by high resolution meiotic mapping. Clones from the BAG library as well as genomic contigs that span the epd locus will be identified. The candidate genes will be tested by gene knock-down using morpholinos to phenocopy the mutation, analyzing the gene mutation by PCR and DNA sequencing, and injecting the cDNA or mRNA to rescue the mutant phenotype. Molecular characterization of the epd gene will enable mechanistic study of its function by future experiments designed to test the hypothesis that epd controls proliferation of exocrine progenitors. These studies can be expected to provide further insights into the genetic regulation of pancreatic organogenesis and tumorigenesis.

描述（由申请人提供）：长期目标是通过了解模型生物中的发育和信号传导机制来理解人类癌症的分子基础来制定预防癌症和治疗的策略。斑马鱼胰腺被用作模型器官，以识别和表征细胞命运和谱系测定，增殖，生存，分化，形态发生和生长的发育调节剂。已经制定了一种策略来确定通过化学诱导的诱变筛查筛查胰腺发育的基因。一（1）个独特的突变体，外分泌和色素沉着（EAP）的外分泌胰腺，正常胰岛和皮肤色素减少。胰管分支是型肌，肌酸分支相对较小，而腺泡细胞则分化，但含有相对较少的Zymogen颗粒。这些数据导致了我们的假设，即EPD突变会导致外分泌胰腺的生长停滞。 EPD基因产物有望在外分泌祖细胞的细胞增殖中起调节作用。这项拟议的研究的目的是通过位置克隆技术鉴定EPD基因。第一个具体目的是基于简单的序列长度多态性来确定EPD基因的染色体位置，并通过高分辨率减数分裂映射定义携带EPD基因的关键区域。将确定来自袋子库的克隆以及跨EPD基因座的基因组重叠群。候选基因将通过使用形态学的基因敲除基因来测试突变，通过PCR和DNA测序分析基因突变，并注入cDNA或mRNA以拯救突变体表型。 EPD基因的分子表征将通过旨在测试EPD控制外分泌祖细胞增殖的假设的未来实验来实现其功能的机械研究。可以预期，这些研究可以进一步见解胰腺机体发生和肿瘤发生的遗传调节。