Pigmented cell lines of mouse albino melanocytes containing a tyrosinase cDNA with an inducible promoter

Abstract

Melanocyte cell lines, with characteristic dendritic morphology and melanosomes, were established from young mice of wild-type (C57 BL/6) and of two albino (C57 BL/6-c ^2J/c ^2J and BALB/c) inbred strains. The albino cells were cotransfected with two plasmids: pMTtyrl, containing the full-length tyrl cDNA for tyrosinase encoded by thec locus, under the control of the inducible mouse metallothionein-I (MT-I) promoter; and pSVneoß, allowing selection of transformants by G418 resistance. The intrinsic albino defect was corrected by the tyrl cDNA in transfected cells, thereby validating the coding capability of tyrl for tyrosinase. Black melanin was formed in the genetically black (B/B) C57 BL/6-c ^2J/c ^2J cells and brown melanin in the genetically brown (b/b) BALB/c cells. Pigment was produced even without adding heavy metal (for induction of the MT-I promoter), thus obviating the need for adding it, but was formed more rapidly upon addition of ZnSO₄ up to 100 µM. Stable transfected albino melanocyte lines with active tyrosinase and melanization were obtained. Addition of ZnSO₄ at 200 µM was lethal to the cells. However, this toxicity—attributable at least in part to melanin precursors—was prevented if the cells sojourned at 100 µM ZnSO₄ for two weeks before being exposed to the 200 µM level. Adaptation was lost when the cells were removed from 200 µM ZnSO₄ for one week and then returned to it. Avoidance of toxicity under these conditions is thus the result of physiological detoxification mechanisms rather than selection for a genetic change.