Department of Biology, Zoophysiology, Aarhus University, Aarhus, Denmark.
Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
Transgenic Res. 2023 Dec;32(6):537-546. doi: 10.1007/s11248-023-00369-3. Epub 2023 Oct 17.
Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.
先前使用肌红蛋白 (Mb) 敲除小鼠和敲低斑马鱼的研究对这种保守且高度表达的蛋白质缺失所导致的体内表型提出了相互矛盾的结果，因此需要一种新的经过良好表征的敲除模型。我们在这里使用 CRISPR/Cas 系统描述了三种不同的斑马鱼 Mb 敲除系的产生。这三种系在胚胎和幼虫发育过程中均未表现出任何形态表型、长度变化或致死性。成年纯合敲除 Mb(Auzf13.2)斑马鱼系缺乏 Mb 蛋白，mb mRNA 几乎完全降解，且在活力、长度或心脏大小方面没有变化。此外，成年心脏组织的转录组分析表明 Mb 敲除不会导致其他基因表达的改变。最后，在 36 个筛选的靶位中没有观察到脱靶。总之，我们生成了三种 Mb 敲除系，在胚胎和幼虫发育期间表现出相似的表型，并验证了其中一种系 mb(Auzf13.2)在成年心脏中没有遗传补偿或脱靶效应的迹象。这些发现表明，mb(Auzf13.2)有望成为研究 Mb 在斑马鱼中的生物学作用的候选基因。
