Department of Biomedical Sciences, College of Medical Convergence, Catholic Kwandong University, Gangneung-si, Gangwon-do, Republic of Korea.
Institute of Reproductive Medicine and Population, Medical Research Center, Dept. of Obstetrics & Gynecology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea.
Stem Cell Res. 2023 Jun;69:103064. doi: 10.1016/j.scr.2023.103064. Epub 2023 Mar 8.
TUBB3 is a structural neuronal protein important for multiple neuronal functions including axonal guidance and maturation. This study aimed to generate a human pluripotent stem cell (hPSC) line with a TUBB3-mCherry reporter using CRISPR/SpCas9 nuclease. The stop codon in the last exon of TUBB3 was replaced with a T2A-mCherry cassette using CRISPR/SpCas9-mediated homologous recombination. The established TUBB3-mCherry knock-in cell line exhibited typical pluripotent characteristics. The mCherry reporter faithfully replicated the endogenous level of TUBB3 upon induction of neuronal differentiation. The reporter cell line could contribute to the investigation of neuronal differentiation, neuronal toxicity, and neuronal tracing.
TUBB3 是一种结构神经元蛋白，对多种神经元功能很重要，包括轴突导向和成熟。本研究旨在使用 CRISPR/SpCas9 核酸酶生成具有 TUBB3-mCherry 报告基因的人多能干细胞 (hPSC) 系。使用 CRISPR/SpCas9 介导的同源重组，将 TUBB3 最后一个外显子中的终止密码子替换为 T2A-mCherry 盒。建立的 TUBB3-mCherry 敲入细胞系表现出典型的多能特征。mCherry 报告基因在诱导神经元分化时忠实地复制了内源性 TUBB3 的水平。该报告细胞系可用于研究神经元分化、神经元毒性和神经元示踪。
