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Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques
Uemura, Takeshi
Mori, Takuma
Kurihara, Taiga
Kawase, Shiori
Koike, Rie
Satoga, Michiru
Cao, Xueshan
Li, Xue
Yanagawa, Toru
Sakurai, Takayuki
Shindo, Takayuki
Tabuchi, Katsuhiko
© The Author(s) 2016. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
Genome editing is a powerful technique for studying gene functions. CRISPR/Cas9-mediated gene knock-in has recently been applied to various cells and organisms. Here, we successfully knocked in an EGFP coding sequence at the site immediately after the first ATG codon of the β-actin gene in neurons in the brain by the combined use of the CRISPR/Cas9 system and in utero electroporation technique, resulting in the expression of the EGFP-tagged β-actin protein in cortical layer 2/3 pyramidal neurons. We detected EGFP fluorescence signals in the soma and neurites of EGFP knock-in neurons. These signals were particularly abundant in the head of dendritic spines, corresponding to the localization of the endogenous β-actin protein. EGFP knock-in neurons showed no detectable changes in spine density and basic electrophysiological properties. In contrast, exogenously overexpressed EGFP-β-actin showed increased spine density and EPSC frequency, and changed resting membrane potential. Thus, our technique provides a potential tool to elucidate the localization of various endogenous proteins in neurons by epitope tagging without altering neuronal and synaptic functions. This technique can be also useful for introducing a specific mutation into genes to study the function of proteins and genomic elements in brain neurons.
Article
SCIENTIFIC REPORTS.6:35861(2016)
NATURE PUBLISHING GROUP
2016-10-26
eng
journal article
VoR
http://hdl.handle.net/10091/00021011
https://soar-ir.repo.nii.ac.jp/records/20253
https://www.ncbi.nlm.nih.gov/pubmed/27782168
27782168
https://doi.org/10.1038/srep35861
10.1038/srep35861
2045-2322
SCIENTIFIC REPORTS
6
35861
https://soar-ir.repo.nii.ac.jp/record/20253/files/srep35861.pdf
application/pdf
2.4 MB
2018-10-31