Chapter title |
CRISPR/Cas9-Mediated In Vitro Mutagenesis in GC-Like B Cells
|
---|---|
Chapter number | 12 |
Book title |
Germinal Centers
|
Published in |
Methods in molecular biology, June 2017
|
DOI | 10.1007/978-1-4939-7095-7_12 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7094-0, 978-1-4939-7095-7
|
Authors |
Chu, Van Trung, Graf, Robin, Rajewsky, Klaus, Van Trung Chu, Robin Graf, Klaus Rajewsky |
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. |
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