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Fig. 4 | BMC Biotechnology

Fig. 4

From: Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

Fig. 4

Cas9 is functional in B cells of Rosa26LSL-Cas9 knock-in mice. a: Scheme of genome editing in primary mouse B cells using CRISPR/Cas9. Naive B cells from spleens of three individual heterozygous Rosa26LSL-Cas9 F1 mice were isolated using CD43 depletion, treated with TAT-Cre and stimulated with LPS for 24 h. TAT-Cre/LPS treated B cells were transduced with retroviral particles co-expressing sgRosa26-1 and BFP to target the Rosa26 locus. One day later, the transduced B cells were selected with puromycin until day 5. b: FACS analysis of B cells (from a) before (day 2) and after puromycin selection (day 5). The gate indicates the fraction (percentage) of successfully transduced BFP+ cells. c: Gel electrophoresis of T7EI or XbaI digested PCR products (R26T7F/R26T7R primers) amplified from DNA of FACS sorted BFP+ cells (from b), indicating sequence deletions by the presence of T7EI sensitive or XbaI resistant bands (arrows)

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