Efficient generation of a CYP3A4-T2A-luciferase knock-in HepaRG subclone and its optimized differentiation
Efficient generation of a CYP3A4-T2A-luciferase knock-in HepaRG subclone and its optimized differentiation
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CYP3A4-T2A-荧光素酶敲入HepaRG亚克隆的高效生成及其优化分化
DOI：
10.1016/j.biopha.2022.113243
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发表时间：
2022-06-07
影响因子：
7.5
通讯作者：
Huang，Lizhen
中科院分区：
文献类型：
--
作者：
Zuo，Qingxia;Xu，Wanqing;Huang，Lizhen
CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-β pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.
