Cell Structure and Function
Online ISSN : 1347-3700
Print ISSN : 0386-7196
ISSN-L : 0386-7196
Knock-in of Labeled Proteins into 5’UTR Enables Highly Efficient Generation of Stable Cell Lines
Faryal IjazKoji Ikegami
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Supplementary material

2021 Volume 46 Issue 1 Pages 21-35


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.

Key words: CRISPR/Cas9, knock-in, primary cilium, UTR, tubulin

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© 2021 by Japan Society for Cell Biology

Copyright: ©2021 The Author(s). This is an open access article distributed under the terms of the Creative Commons BY (Attribution) License (https://creativecommons.org/licenses/by/4.0/legalcode), which permits the unrestricted distribution, reproduction and use of the article provided the original source and authors are credited.
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