Fig. 3
From: Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes
Cas9 is expressed in B cells of Rosa26LSL-Cas9 knock-in mice. a: Strategy for isolating naive B cells from spleens of three Rosa26LSL-Cas9 F1 mice by CD43 depletion and activation of Cas9 expression by deletion of the loxP flanked stop element upon treatment with TAT-Cre protein. b: Scheme of the TAT-Cre-mediated deletion of the Neo/Stop element (left). Detection of Cre-mediated deletion of the Neo/STOP cassette by PCR using DNA of TAT-Cre/LPS-treated B cells and the indicated primers. Rosa26LSL-Cas9 alleles produce a 1.0 kb band (CAGF-NeoR1 primers), Cre recombined alleles are detected by a 0.7 kb band (CAGF/Cas9R1 primers). c: Sequencing results of 0.7 kb PCR products (from b) showing the correct deletion of the loxP flanked stop element, leaving one loxP site in between the CAG promoter and the Cas9 coding region. d: Western blot analysis of lysates prepared from TAT-Cre/LPS treated B cells of three Rosa26LSL-Cas9 F1 mice using antibodies against the Flag Tag, Cas9, or Beta-actin