Preservation of phenotype in an organotypic cell culture model of a recessive keratinization defect of Norfolk terrier dogs

Abstract

The purpose of this study is to reproduce in vitro a recessive keratinization defect of Norfolk terrier dogs characterized by a lack of keratin 10 (K10) production. Keratinocytes from skin biopsy samples of four normal dogs and two affected dogs were cultured organotypically with growth factor-supplemented media in order to stimulate cornification. The cultured epidermis from the normal dogs closely resembled the normal epidermis in vivo and cornified. The cultured epidermis from the affected dogs displayed many phenotypic alterations identified in skin biopsies from dogs with this heritable defect. Immunohistochemistry and immunoblotting showed a marked decrease in K10 from the cultures of the affected keratinocytes, compared to that in K10 from the cultures of the normal keratinocytes. Real-time reverse transcription polymerase chain reaction quantitation showed a 31-fold decrease in K10, a 1.75-fold increase in K1 and a 136-fold increase in K2e between the affected and the normal epidermis. Organotypic keratinocytes showed a 241-fold decrease in K10, a 31-fold decrease in K1 and a 1467-fold decrease in K2e between the affected and normal cultures. Although in vitro keratin expression did not precisely simulate in vivo, the morphology of the normal and the affected epidermis was largely preserved; thus, this culture system may provide an alternative to in vivo investigations for cutaneous research involving cornification.