Figure 2

Evaluation of KI efficiency for an inactive gene. (a) Schematic diagram of the PLP1-EGFP construct. The PLP1-EGFP TV harboured 3.1-kb and 5.0-kb homology arms, which consisted of the region surrounding the initial codon of the marmoset PLP1 gene locus. EGFP coding sequence was fused to the 3′ end of the 5′-UTR of the PLP1 gene. Polyadenylation signal sequence (pA) was introduced downstream of the terminal codon of EGFP. Additionally, loxP-flanked PGK-Neo-pA was placed between EGFP-pA and the 3′-homology arm. The TV was not detected by gRNAs targeting the vicinity of PLP1 exon 1 (PLP-1, 2, 3, 4, shown as scissors). Thin black arrows indicate the location of the 5′-external primer and the 3′-internal primer used for genotyping PCR of the 5′-region. Grey arrows indicate the 5′-primer homologous to the selection cassette and the 3′-external primer used for genotyping PCR of the 3′-region; b, a restriction enzyme site (BglII); p, the 5′-external probe for Southern blotting. (b) The number of G418-resistant colonies following G418 selection of 1 × 10⁶ transfected cjESCs, shown as the mean + s.e.m., n = 3. (c) 5′-region genotyping PCR of G418-resistant cjESC clones. Cas9+ clone numbers are shown in grey (#17–22), and Cas9− are shown in black (#17–22). Results for the remaining clones are shown in Supplementary Fig. S4a. Ho, homozygous KI; He, heterozygous KI. (d) Southern blotting analysis of G418-resistant Cas9− clones using the 5′-external probe. (e) Summary of the PLP1 exon 1 gene targeting data. *P < 0.05, **P < 0.01.