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Fig. 2. Generation of Arnt-deficient mice. (A) Domain structure of Arnt. NLS, nuclear localization signal; bHLH, basic helix-loop-helix domain; PAS, Per-Arnt-Sim domain; TAD, transcription activation domain. (B) Generation of a K14-driven Arnt-deficient mouse model. LoxP sites flank exon 6 of the Arnt gene (see Tomita et al., 2000). Activity of Cre recombinase is introduced by crossbreeding of Arnt-floxed and K14-Cre animals, resulting in the excision of the exon 6 encoding the bHLH domain. (C) PCR analysis of the progeny obtained from crossbreeding of Arnt^flox/flox and K14-Cre mice. Lane 1, Arnt wild-type allele; lane 2, Arnt^flox/-:K14-Cre^-; lane 3, Arnt^floxflox:14-Cre^-; lane 4, Arnt^flox/-:K14-Cre⁺, deleted () band is present; lane 5, Arnt^flox/flox:K14-Cre⁺, deleted () band is present. The presence of the undeleted (floxed) bands in lanes 4 and 5 is explained by contamination of the sample with genomic DNA from K14-negative tissues (e.g. cartilage or dermis). (D) Western blot performed with proteins isolated from epidermis of Arnt^/ pups and cultured Arnt^/ keratinocytes shows absence of Arnt protein, thus confirming the efficiency of gene targeting in our mouse model.

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