CRISPR/Cas9-Mediated In Vitro Mutagenesis in GC-Like B Cells.
Methods Mol Biol
; 1623: 135-145, 2017.
Article
em En
| MEDLINE
| ID: mdl-28589354
ABSTRACT
The CRISPR/Cas9 technology has developed into a powerful tool for genome editing, both in terms of gene silencing and the insertion of precise mutations. However, the application of CRISPR/Cas9-mediated mutagenesis in primary immune cells, in particular in B cells, is still in its infancy because of the difficulty to deliver the CRISPR/Cas9 system into these cells. Here, we describe a new method to use CRISPR/Cas9 for manipulating genes in germinal center (GC)-like B cells in vitro. We isolated Cas9-expressing B cells from R26-Cas9iGFP/+ mice (expressing Cas9 constitutively from the Rosa26 locus) and mixed them with control B cells. Primary B cells were cultured on CD40L- and BAFF-expressing feeder cells and transduced with retroviral particles expressing the sgRNAs of interest. Using this system, we have achieved complete gene knockouts in up to 92% of activated B cells.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Linfócitos B
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Mutagênese
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Marcação de Genes
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Centro Germinativo
/
Sistemas CRISPR-Cas
Limite:
Animals
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Alemanha