Claudin 6 structure, distribution and transgenic phenotype. Claudin 6 structure, distribution and transgenic phenotype. (A) Molecular structure analysis of claudin 6 predicts it to be a 4 trans-membrane domain integral membrane protein that has varying tissue distribution in newborn mice. (B) Expression of claudin 6 was detected strongly in liver and kidney while there were varying degrees of expression in heart, thymus, spleen, calveria, brain, lung, tongue, stomach, tail and skin. There was no detectable expression in intestine. (C) RT-PCR results were supported by immunohistochemical staining of wild-type sections of intestine, lung and kidney tissues. (D) Mouse claudin 6 was cloned into the NotI site of an expression vector containing the human involucrin promoter/enhancer and 17 transgenic animals were generated. (E) PCR analysis was used to detect expression of the claudin 6 transgene using primers spanning the involucrin promoter to the claudin 6 coding sequence (Table 1). A 590 bp band was detected in lane 2 (plasmid DNA control) as well as in lanes 6-10 (positive transgenic mice). There was no band detectable in lane 1 (PCR control) and lanes 3-5 (non-transgenic mice). (F) RT-PCR was also performed using primers spanning the exon sequences present in the involucrin promoter to the claudin 6 coding sequence in order to identify only the transgene expression (692 bp). (G) claudin 6 forward and reverse primers also identified the endogenous expression (660 bp), which is approximately 8-fold less than that of the transgenic expression. Western blotting of proteins extracted from the epidermis of transgenic and wild-type mice with claudin 6 antibodies revealed a 2.5-fold increase in the transgenic samples over the wild-type expression (H). The increased transgene expression was also demonstrated through indirect immunofluorescence with a marked increase of claudin 6 protein detected in the upper spinous and granular layers of transgenic backskin samples as compared to their wild-type counterparts (I). Kursad Turksen, and Tammy-Claire Troy Development 2002;129:1775-1784 © 2002.