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标记突变体实现斑马鱼仅凭视觉观察的快速非侵入性基因分型

Marked Mutants Allow for Rapid Non-Invasive Genotyping of Zebrafish by Visual Inspection Alone

Europe PMC · 预印本(领域内) · bioRxiv · 预印本 · 2026-11-03
Juhasz SC, Shaw-McGrath T, Keatinge M
DOI 10.64898/2026.10.29.762891 · 原文链接 ↗ · Scholar
综合 65.0 模型 60.0 · deepseek/deepseek-flash · 强度 low 已入选 动物模型与比较生殖 生殖干细胞与体外配子发生 伦理、法规与指南
★ 命中偏好:可转化到 ART 实验室的发现 (加权 +5.0,仅影响排序与入选,不是收录条件)
新颖 80严谨 70影响 55领域相关 30转化 35

摘要

该预印本在斑马鱼中利用CRISPR敲入策略,将包含启动子、荧光蛋白和polyA的完整次级标记盒插入目标基因编码外显子,使靶基因功能被破坏的同时永久标记突变等位基因,称为“标记突变体”。研究针对mitfa、tdp43和tdpl构建8个独立等位基因,使用孵化腺、胰腺或松果体启动子驱动GFP/RFP/BFP,发现生殖系传递效率约30%,所有等位基因均表现功能缺失;组合四个不同荧光标记可在表型出现前仅凭视觉识别tdp43/tdpl双复合杂合胚胎。该方法可减少PCR和剪鳍,简化群体管理并促进组合遗传学。

原文摘要

Genetic analysis in zebrafish relies heavily on invasive, labour-intensive molecular genotyping. This limits throughput and phenotypic analysis, particularly for homozygous mutants that lack readily detectable phenotypes at early developmental stages. To overcome these limitations, we adapted CRISPR-mediated knock-in strategies to insert complete secondary marker cassettes (including promoter, fluorophore and polyA), directly into coding exons of target loci. This simultaneously disrupts target gene function and permanently labels mutant alleles, which we term Marked Mutants. Crossing heterozygous Marked Mutant alleles targeting the same gene but carrying distinct fluorescent secondary markers linked to each allele, enables unambiguous non-invasive visual identification of wild-type, heterozygous and biallelic mutant progeny as early as 24 hours post fertilisation. To test the scalability and robustness of our approach, we generated eight independent Marked Mutant alleles targeting mitfa, tdp43 and tdpl, using secondary markers comprising hatching gland (he1.1), pancreas (ins) or pineal gland (exorh) promoters driving expression of GFP, RFP or BFP. We found germline transmission efficiencies comparable to conventional CRISPR mutagenesis, with approximately 30% of fluorescence-selected F0 adults transmitted on-target insertions to the F1 generation. All Marked Mutant alleles behaved as loss-of-function alleles, demonstrating effective disruption of target gene function. By combining four independent Marked Mutant alleles, each carrying a distinct fluorescent secondary marker, we could prospectively identify tdp43/tdpl double compound heterozygous embryos by visual inspection alone before phenotypic onset. Only embryos carrying all four Marked Mutant alleles developed the characteristic lethal double-knockout phenotype, while siblings carrying fewer alleles remained phenotypically normal. By eliminating routine PCR genotyping and invasive fin clipping, Marked Mutants simplify colony management, facilitate combinatorial genetics, reduce animal use, and provide a practical platform for rapid non-invasive zebrafish reverse genetics.

推荐理由:方法学新颖,对斑马鱼反向遗传学和动物模型管理有实用价值;但与人类生殖医学/ART核心问题距离较远,主要适合从事动物模型与发育遗传学的研究者浏览。

关键词:斑马鱼 · CRISPR敲入 · 非侵入性基因分型 · 标记突变体 · 荧光报告 · 动物模型

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文献解读

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所属事件:标记突变体实现斑马鱼仅凭视觉观察的快速非侵入性基因分型 热度 4.1 · 1 个来源

条目 #437 · 入库 2026-11-08 11:44 · JSON · Preprint