Generation of Maternal Mutants Using <em>zpc:cas9</em> Knock-in Zebrafish

Yizhuang Zhang; Ziping Fu; Boya Yang; Jiasheng Wang; Tong Lu; De-Li Shi; Ming Shao

doi:10.3791/68642

JoVE Journal
Developmental Biology

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JoVE Journal Developmental Biology

Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish

使用 zpc：cas9 敲入斑马鱼生成母系突变体

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09:17 min

July 22, 2025

DOI: 10.3791/68642-v

Yizhuang Zhang¹, Ziping Fu¹, Boya Yang¹, Jiasheng Wang¹, Tong Lu¹, De-Li Shi^2,3, Ming Shao^1,4,5,6

¹Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences Qilu Hospital (Qingdao), Cheeloo College of Medicine,Shandong University, ²Institut de Biologie Paris-Seine (IBPS), UMR CNRS 8263, INSERM U1345, Development, Adaptation and Ageing,Sorbonne Université, ³College of Marine Life Sciences,Ocean University of China, ⁴Key Laboratory for Experimental Teratology of the Ministry of Education,Shandong University, ⁵State Key Laboratory of Microbial Technology,Shandong University, ⁶Shandong University-Yuanchen Joint Biomedical Technology Laboratory

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在这里，我们描述了一种产生母系突变体的方案，该方案将稳定的 zpc：cas9 敲入线与 Tol2 介导的 sgRNA 表达盒的递送偶联。

在本视频中，我们介绍了一种改进的斑马鱼卵母细胞特异性条件敲除方法，为研究具有合子突变导致致死或不育的母体因素提供了一个多功能平台。目前的母体敲除方法要么在技术上具有挑战性，要么非常耗时。我们之前报道过 zpc：cas9 转基因在几代人中被转录沉默。

使用 zpc：cas9 敲入系，我们建立了一个强大的平台，用于在卵子发生过程中产生条件敲除，从而能够快速、高效地生产母体突变体。首先，收集由纯合子 rbm24a RFPKI zpc：cas9 鱼产卵的胚胎。将一微升 PGG 粉尘 EB rbm24a 4sGRNA 与一微升 Tol2 转座酶信使 RNA 混合以制备注射混合物。

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