Targeted Cellular Ablation in Transgenic Zebrafish

描述(申请人提供)：随着人类年龄中位数的增加，退行性疾病已成为主要的健康问题。该项目的长期目标是确定促进特定细胞类型再生的药物，从而逆转衰弱退行性疾病的影响。我们开发了一种可诱导的斑马鱼靶向细胞消融方法，称为ZAP(斑马鱼消融报告蛋白)，它将促进高通量方法：1)定义调节与特定退行性疾病相关的细胞类型再生的基因；2)在退行性疾病的突变模型中识别促进细胞类型特异性再生的药物。ZAP转基因平台为揭示脊椎动物系统中细胞再生的“基因组学”提供了第一个机会，而且它本身就是可许可的，因为它可以应用于任何与离散细胞丧失有关的退行性疾病状态。此外，突变模型将疾病与离散的分子靶点联系起来，减少了药物发现的时间和成本。具体目的有：1.瞬时转基因斑马鱼中ZAP的嵌合表达和对照报道。当共注射时，将组装在瞬时转基因斑马鱼胚胎和幼体中重叠和非重叠的细胞亚群中表达ZAP和对照报告蛋白的质粒组。2.对表达ZAP的斑马鱼进行靶向细胞消融：剂量、特异性、有效性。共表达ZAP和对照质粒的瞬时转基因斑马鱼将被用于测试特定消融诱导剂的特性以及单个细胞和组织类型的响应性。在记录了表达模式后，将添加试剂来诱导ZAP表达细胞的消融。特异性将通过监测ZAP连锁和对照报告的表达来评估。将针对单个消融诱导剂确定靶向消融与局部消融效果的有效前药物浓度，以及适用于高通量筛选分析的方案。3.建立稳定的转基因斑马鱼品系，在靶细胞类型中表达ZAPS。将创建由可操作地连接到ZAP表达序列的细胞型特定启动子元件组成的质粒。从这些质粒的稳定整合中获得的转基因株将作为帕金森氏症等退行性疾病的模型。

DESCRIPTION (provided by applicant): Degenerative diseases have become major health issues as the median age of humans has increased. The long-term goal of this project is to identify drugs, which promote the regeneration of specific cell types, thus reversing the effects of debilitating degenerative conditions. We have developed an inducible method of targeted cellular ablation in zebrafish, termed ZAP (zebrafish ablation-reporter protein), that will facilitate high-throughput approaches for: 1) Defining genes which regulate the regeneration of cell types relevant to specific degenerative conditions, and; 2) Identifying drugs which promote cell-type specific regeneration in mutational models of degenerative diseases. The ZAP transgenic platform provides the first opportunity to reveal the "genomics" of cellular regeneration in a vertebrate system and is inherently licensable as it can be applied to any degenerative disease state linked to the loss of discrete cells. Furthermore, mutational models link diseases to discrete molecular targets, reducing the time and cost of drug discovery. Specific aims are: 1. Mosaic expression of ZAP and control reporters in transient transgenic zebrafish. Plasmids will be assembled that, when co-injected, express ZAP and control reporter proteins in overlapping and non-overlapping subsets of cells in transient transgenic zebrafish embryos and larvae. 2. Targeted cellular ablation in ZAP-expressing zebrafish: Dosage, Specificity, Effectiveness. Transient transgenic zebrafish co-expressing ZAP and control plasmids will be used to test the characteristics of specific ablation-inducing agents and the responsiveness of individual cell and tissue types. After documenting expression patterns, reagents will be added to induce the ablation of ZAP expressing cells. Specificity will be assessed by monitoring ZAP-linked and control reporter expression. Effective pro-drug concentrations for targeted versus regional ablation effects, and protocols appropriate for high-throughput screening assays will be determined for individual ablation-inducing agents. 3. Create stable transgenic zebrafish lines expressing ZAPs in targeted cell types. Plasmids will be created that consist of cell-type specific promoter elements operably linked to ZAP expression sequences. Transgenic lines derived from the stable integration of these plasmids will serve as models of degenerative diseases such as Parkinson's disease.