Generation of PEPCK-C-/- mice. Panel A. Diagram of the targeting vector, which is aligned with endogenous sequence of PEPCK-C gene. The sequence between Pst I and Xho I, which covers part of the gene promoter and the exons 1 and 2 in endogenous gene, was replaced with Neo resistance gene (PGK_βgal-neoR) in the targeting vector. H, Hind III; P, Pst I; X, Xho I. PGK_βgal-neoR, the phosphoglycerate kinase gene promoter drives a fusion gene of βgal and neoR. DTA, the diphtheria toxin A chain. Panel B. Genotyping of the PEPCK-C-/- mice by Southern blotting. Genomic DNA was digested with Hind III and hybridized with a PEPCK-C cDNA probe composed of exons 1~6 of the rat PEPCK-C gene (black bars). For the wild type allele of PEPCK-C, 1.3-kb and 2.75-kb fragments were detected; only one fragment (2.5 kb) was excepted for the targeted allele.