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

Abstract：

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. RESULTS:Dissociated 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. CONCLUSIONS:Detection 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.

journal_name

BMC Neurosci

journal_title

BMC neuroscience

authors

Tsunematsu H,Uyeda A,Yamamoto N,Sugo N

doi

10.1186/s12868-017-0377-9

subject

Has Abstract

pub_date

2017-08-01 00:00:00

pages

55

issue

1

issn

1471-2202

pii

10.1186/s12868-017-0377-9

journal_volume

18

pub_type

杂志文章

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