Knock-in of Labeled Proteins into 5'UTR Enables Highly Efficient Generation of Stable Cell Lines.
Knock-in of Labeled Proteins into 5'UTR Enables Highly Efficient Generation of Stable Cell Lines.
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DOI：
10.1247/csf.21002
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发表时间：
2021-03-16
影响因子：
1.5
通讯作者：
中科院分区：
文献类型：
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表达标记蛋白的稳定细胞系和动物模型是研究细胞和分子行为的重要工具。几种分子生物学技术已经以不同程度的成功和效率应用于建立表达标记蛋白的细胞系。在这里，我们应用CRISPR/Cas9将标记蛋白敲入内源基因座的5 'UTR。利用这种5 'UTR靶向敲入策略，在抗生素选择的细胞中以50至80%的高效率建立了表达Ar 113 b-Venus、Reep 6-HA和EGFP-α-微管蛋白的稳定细胞系。敲入蛋白的定位与野生型细胞中的内源蛋白的定位相同，并且显示出同源表达。此外，从内源性启动子敲入的EGFP-α-微管蛋白的表达在长期培养中是稳定的。我们进一步证明了荧光信号对于长时间的延时成像是足够的。在整个延时成像期间，荧光信号清晰可见，并显示出特定的亚细胞定位。总之，我们的策略表明，5 'UTR是一个顺从的网站，以产生细胞系的稳定表达的标记蛋白质从内源基因座在哺乳动物细胞。
Stable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biology technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5’UTR of the endogenous gene loci. With this 5’UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high efficiencies ranging from 50 to 80% in antibiotic selected cells. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5’UTR is an amenable site to generate cell lines for the stable expression of tagged proteins from endogenous loci in mammalian cells.
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影响因子：
3.3
作者：
Katoh Y;Michisaka S;Nozaki S;Funabashi T;Hirano T;Takei R;Nakayama K
通讯作者：
Nakayama K
影响因子：
7
作者：
Auer TO;Duroure K;De Cian A;Concordet JP;Del Bene F
通讯作者：
Del Bene F
DOI：
10.1073/pnas.96.9.5037
发表时间：
1999-04-27
影响因子：
11.1
作者：
Mao, XH;Fujiwara, Y;Orkin, SH
通讯作者：
Orkin, SH
影响因子：
7
作者：
Maresca M;Lin VG;Guo N;Yang Y
通讯作者：
Yang Y
影响因子：
46.9
作者：
Kuscu, Cem;Arslan, Sevki;Adli, Mazhar
通讯作者：
Adli, Mazhar