Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques
抄録
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)
収録刊行物
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- Scientific Reports
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Scientific Reports 6 35861-, 2016-10
Nature Publishing Group
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詳細情報 詳細情報について
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- CRID
- 1050282677625225856
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- NII論文ID
- 120007100133
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- ISSN
- 20452322
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- HANDLE
- 2241/00144431
- 10091/00021011
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- 本文言語コード
- en
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- 資料種別
- journal article
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- データソース種別
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- IRDB
- CiNii Articles