0 citations0 references

Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques

Scientific Reports2016Vol. 6(1), pp. 35861–35861

Citations Over TimeTop 10% of 2016 papers

Takeshi Uemura, Takuma Mori, Taiga Kurihara, Shiori Kawase, Rie Koike, Michiru Satoga, Xueshan Cao, Xue Li, Toru Yanagawa, Takayuki Sakurai, Takayuki Shindo, Katsuhiko Tabuchi

Abstract

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.

Citations Over TimeTop 10% of 2016 papers

Abstract

Related Papers