斑马鱼在功能基因组学和疾病建模中的使用由于易于用基因组编辑核酸酶进行靶向诱变而变得流行,即,锌指核酸酶(ZFN),转录激活因子样效应核酸酶(TALEN),并聚集规则间隔的短回文重复序列/Cas9(CRISPR/Cas9)。这些核酸酶,特别地,CRISPR/Cas9通常用于通过在靶基因中的期望位点处引起双链断裂并选择由修复过程中的错误引起的移码插入或缺失(indel)来产生基因敲除突变体。因此,在突变体产生和表型分析的过程中,已经开发了多种方法来鉴定带有indel的鱼类。这些方法的范围从PCR和基于凝胶的低通量方法到需要特定试剂和/或设备的高通量方法。这里,我们对斑马鱼目前常用的indel检测方法进行了全面综述。通过讨论每种方法的分子基础及其优缺点,我们希望这篇综述将成为斑马鱼研究人员的综合资源,允许他们根据预算选择最合适的方法,获得所需设备和项目的吞吐量需求。
The use of zebrafish in functional genomics and disease modeling has become popular due to the ease of targeted mutagenesis with genome editing nucleases, i.e., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These nucleases, specifically CRISPR/Cas9, are routinely used to generate gene knockout mutants by causing a double stranded break at the desired site in the target gene and selecting for frameshift insertions or deletions (indels) caused by the errors during the repair process. Thus, a variety of methods have been developed to identify fish with indels during the process of mutant generation and phenotypic analysis. These methods range from PCR and gel-based low-throughput methods to high-throughput methods requiring specific reagents and/or equipment. Here, we provide a comprehensive
review of currently used indel detection methods in zebrafish. By discussing the molecular basis for each method as well as their pros and cons, we hope that this
review will serve as a comprehensive resource for zebrafish researchers, allowing them to choose the most appropriate method depending upon their budget, access to required equipment and the throughput needs of the projects.