Fig. S4.

Strategy for targeted deletion of hcIE in ES cells. (A) WT allele: genomic organization and a partial restriction map of the Il4 locus, including hcIE. Targeting construct: a 445-bp segment that included hcIE was replaced by an loxP-flanked neo. Mutated locus after Cre recombination: Cre-mediated excision of the neo cassette in the germ line resulted in the final arrangement. DT-A, diphtheria toxin A chain expression cassette; E1–E4, four exons of the Il4 gene; H, HindIII; P, PstI; S, SmaI; triangles, loxP sites. (B) Southern blot analysis to ensure correct integration of the targeting construct in hcIE-KO mice. Genomic DNA was prepared from mouse tail biopsies, digested with PstI and HindIII, separated by electrophoresis, and hybridized with the indicated probe. The WT and targeted alleles yielded 5.6- and 2.0-kbp fragments, respectively. (C) PCR analysis of Cre-mediated neo cassette excision from the targeting construct in hcIE-KO mice on the CD4-Cre-Tg background. Cre-mediated excision from genomic DNA was confirmed by PCR. Primers were designed for the 3′- and 5′-flanking regions of the targeted locus and are indicated by arrows. The WT, targeted, and mutated alleles yielded 508-, 1,426-, and 127-bp fragments, respectively.