Fig. 1

Generation of intersectional Rosa26 mouse lines. A Targeting schematic showing the modular targeting vector containing a 1-kb 5′ homology arm, CAG promoter, FRT-flanked neomycin and stop cassette, LoxP-flanked (optional) subtractive gene of interest (GOI-S) and stop cassette, intersectional gene of interest (GOI-I), WPRE, BGHpA element, and 1.2-kb 3′ homology arm. The full intersectional cassette was knocked into the Rosa26 locus, with a 22-bp deletion of the CRISPR sgRNA. B PCR genotyping of neomycin selected ES cell clones. Targeting knock-in was determined using PCR primers that spanned from outside the Rosa26 homology arms to either the CAG promoter (5′ end) or WPRE (3′ end). Amplification of a band indicates targeting. Shown are results from a targeting event with over 60% targeting efficiency (RR7). C Four conditions with different Cas9:targeting vector ratios were used in our initial study: a 0:1 with no Cas9, 0.5:1, 1:1, and 10:1. D Targeting efficiency results from the different ratios. The 10:1 Cas9:targeting vector ratio showed a 5–10-fold increase over an electroporation with no Cas9. Shown in red are results from previous electroporations using the traditional Rosa26 targeting vector with more commonly used longer homology arms