Fig. 1

Generation of CaMK1 floxed mice. A Diagram of the constructs. The gRNAs and the ssODNs were designed to introduce a 5’-side LoxP (5’ LoxP) into the intron upstream of exon 5, and a 3’-side LoxP (3’ LoxP) into the intron downstream of exon 6. The distance between the LoxPs is 1.7 kb. B Scheme of sequential in vitro fertilization and genome editing over two generations. C Genotyping PCR of ten mice born after two rounds of genome editing. Arrowheads indicate target bands. D DNA sequencing around the LoxPs of CaMK1 floxed mouse (#1164). The upper panel is around 5’ LoxP, and the lower panel is around 3’ LoxP. No deletion or mismatch could be identified. E Detailed results of the first and second rounds of genome editing. F in vitro Cre protein treatment of the PCR product from CaMK1 floxed mouse (#1164) genome. pLox2+, a 3.6 kb plasmid with two LoxPs, was used as a positive control. A littermate (#1163) with two LoxPs on different alleles was used as a negative control. Arrowheads indicate the excised DNA. The expected size of the excised DNA is 2787 bp for pLox2+, and the rest is 838 bp. In CaMK1 floxed mice, the expected size of the excised DNA is 1772 bp, and the rest is 527 bp. +; Cre protein-treated group, −; group not treated with Cre protein