Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures

BMC Neuroscience

Methodology Article

Hiroto Tsunematsu¹  Noriyuki Sugo¹  Akiko Uyeda¹  Nobuhiko Yamamoto¹ 

[1] Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, 565-0871, Osaka, Japan;

关键词: CREB;    FOS;    CRISPR/Cas9;    Cortical neuron;    Gene disruption;    Immunocytochemistry;   

DOI  :  10.1186/s12868-017-0377-9

 received in 2016-12-29, accepted in 2017-07-25,  发布年份 2017

来源: Springer

【 摘 要 】

Background CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging.ResultsDissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity.ConclusionsDetection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

【 授权许可】

CC BY   
© The Author(s) 2017