Generation of TSC-22–deficient mice. (A) Schematic representation of the targeting construct and homologous recombination in the TSC-22 region. Restriction enzyme sites of KpnI, NheI, and XbaI at the TSC-22 locus are indicated as K, N, and X, respectively. The probes used in Southern screening of embryonic stem cell and the expected sizes of endogenous and mutated fragments obtained by KpnI single digestion or XbaI and NheI double digestion are shown. (B) Southern blot analysis of KpnI-digested DNA extracted from the littermates derived from a cross between heterozygous TSC-22 mice by using probe 1 (A). The 7.5-kb band indicates the existence of the TSC-22 allele, whereas the 4.0-kb band shows the allele loss. The double digestion of XbaI and NheI followed by hybridization with probe 2 (A) gives rise to a 9.5-kb fragment for the existence of the TSC-22 allele and a 7.5-kb fragment for the allele loss (not shown). (C) Northern blotting (left panel) and Western blotting (right panel) to confirm the lack of TSC-22 expression in the TSC-22^−/− mice.