Conditional Atg7 Knockout Mice Generation

The targeting vector was constructed by insertion of a loxP sequences within introns 13 and 14 of Atg7 gene. Exon 14 was fused to a cDNA fragment encoded by exons 15, 16, and 17 (aa 1786–2097) and polyA signal sequence was added after the stop codon. Neo resistant gene cassette (mc1-neo-pA) was ligated behind the polyA signal sequence followed by the second loxP sequence, splicing acceptor site, and exon 14 with stop codon preceding the active site. We electroporated the targeting vector into mouse TT2 ES cells, selected with 200 μg/ml G418 (GIBCO BRL), and then screened for homologous recombinants by PCR and Southern blot analyses. PCR primers were as follows: 5′-TGGCTGCTACTTCTGCAATGATGT-3′, 5′-GAAGGGACTGGCTGCTATTGGGCGAAGTGC-3′, and 5′-TTAGCACAGGGAACAGCGCTCATGG-3′. Southern blot analysis was performed by digestion of genomic DNA with EcoRV and hybridization with the probe shown in Fig. 1 A. Genotyping of mice by PCR was performed using the following two primers: 5′-TGGCTGCTACTTCTGCAATGATGT-3′ and 5′-CAGGACAGAGACCATCAGCTCCAC-3′. Progeny containing the Atg7^Flox allele were bred with Zp3-Cre and Mx1-Cre transgenic mice to produce Atg7^−/− and Atg7^F/F:Mx1 mice, respectively. With regard to Atg7^F/F:Mx1 mice, Cre expression in the liver was induced by i.p. injection of pIpC (Sigma-Aldrich). 300 μl pIpC solution (1 mg/ml in water) was injected three times at 48-h intervals. Mice were housed in specific pathogen-free facilities, and the experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of the Tokyo Metropolitan Institute of Medical Science.