A Knock-in Zebrafish Reporter Line for Live Visualization of Endogenous Olig2 Protein Dynamics.

IF 1.2
Zebrafish Pub Date : 2025-10-01 Epub Date: 2025-09-24 DOI:10.1177/15458547251376166
Chia-Teng Chang, Toru Kawanishi, Sandy Nandagopal, Sean G Megason, Tony Y-C Tsai
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Abstract

The transcription factor oligodendrocyte transcription factor 2 (Olig2) plays a central role in specifying motor neurons and oligodendrocytes during vertebrate neural development. While transgenic reporter lines such as TgBAC(olig2:EGFP) have been instrumental in visualizing olig2 expression, they fall short in directly reporting endogenous protein levels and may not fully recapitulate native gene regulation. To address these limitations, we generated a TgKI(olig2-mNeonGreen) zebrafish line using CRISPR/Cas9-mediated knock-in at the endogenous olig2 locus. The resulting Olig2-mNeonGreen fusion protein localizes specifically to the nucleus, enabling direct live imaging and accurate quantification of Olig2-expressing cells. We confirmed that the knock-in preserves endogenous mRNA expression and protein function, and that homozygous fish develop normally. As proof of concept, modulation of Sonic Hedgehog signaling altered Olig2-mNeonGreen+ cell numbers as expected, confirming the reporter's responsiveness to known upstream inputs. This TgKI(olig2-mNeonGreen) line offers a robust tool for studying neural progenitor dynamics in vivo.

内源性寡聚2蛋白动态可视化的敲入斑马鱼报告线。
转录因子少突胶质细胞转录因子2 (Olig2)在脊椎动物神经发育过程中对运动神经元和少突胶质细胞起着核心作用。虽然TgBAC(olig2:EGFP)等转基因报告系在可视化olig2表达方面发挥了重要作用,但它们在直接报告内源性蛋白水平方面存在不足,并且可能无法完全概括天然基因调控。为了解决这些局限性,我们使用CRISPR/ cas9介导的内源性olig2位点敲入,生成了TgKI(olig2- mneongreen)斑马鱼系。由此产生的Olig2-mNeonGreen融合蛋白特异性定位于细胞核,可以直接对表达olig2的细胞进行实时成像和准确定量。我们证实敲入保留了内源性mRNA表达和蛋白质功能,并且纯合子鱼正常发育。作为概念的证明,Sonic Hedgehog信号的调制如预期的那样改变了Olig2-mNeonGreen+细胞数量,证实了报告者对已知上游输入的响应性。TgKI(olig2-mNeonGreen)系为研究体内神经祖细胞动力学提供了一个强大的工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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