Fig. 2

Generation of floxed alleles for Ambra1, Col12a1, and Ubr5 using Easi-CRISPR. a–c The wild-type alleles, floxing ssDNA donors for targeting exons 4, 2, and 58 of Ambra1, Col12a1, and Ubr5, respectively, and the corresponding floxed alleles. The lengths of ssDNA, homology arms, and the distance between the two LoxP sites are shown. d–f The primer pairs and genotyping PCRs are indicated as in Fig. 1f. The floxed allele schematics show minor differences in primer locations for each gene with respect to target exon size and location. g–i Genotyping of G0 offspring. The expected sizes of PCR amplicons (wild type or floxed) are indicated to the left of the gels. j–l Genotype interpretations are summarized below the gel images (M monoallelic, B biallelic, N no insertion). j Interpretation of Ambra1 genotyping: animals 1, 2, 3, 5, 7, and 8 had both the 5′ and 3′ LoxP sites located in cis. Note that animals 4 and 6 also contain additional amplicons smaller than the expected size (shown by arrows), suggesting that they harbor deletions and/or are mosaic. The sequences of the deletion alleles were not determined. Animals 1 and 5 were bred to wild type and a CD4 Cre mouse line (Fig. 5; Additional file 1: Figure S4). k Interpretation of Col12a1 genotyping: animals 1 and 2 were heterozygous for both 5′ and 3′ LoxP sites located in cis, and they carried deletions in their second allele (shown by the arrows); animal 3 was biallelic for both the 5′ and 3′ LoxP sites. The lanes between the marker and the samples in the full-length PCR gel image (bottom panel) were cropped out because they belonged to another experiment. l Interpretation of Ubr5 genotyping: animals 1 and 2 were heterozygous for both 5′ and 3′ LoxP sites located in cis