Transient expression of a transfected gene in cultured epidermal keratinocytes: implications for future studies.
We examine the effect of keratinocyte differentiation upon transient expression of a nonepithelial gene following DNA-mediated transfer. Cultures of primary epidermal keratinocytes were transfected with the reporter gene, chloramphenicol acetyltransferase (CAT). The CAT gene was linked at the 5' end to the long terminal repeat (LTR) regulatory sequences from Rous sarcoma virus, and gene transfer was accomplished by the calcium phosphate coprecipitation method. Transfected cells were fractionated on Ficoll 400 density gradients. The major finding of this study was that the larger, more differentiated cells displayed five- to seven-fold higher levels of CAT activity per cell than the smaller, less differentiated cells. The higher levels of CAT activity did not result from greater uptake of DNA because cells of all gradient fractions contained one to two copies of plasmid DNA per cell. Furthermore, the CAT gene linked to the regulatory sequences from another virus, SV40, gave the same result. We conclude that the CAT gene, when controlled by these viral regulatory sequences, is expressed more efficiently in differentiated keratinocytes. These results have important implications for the interpretation of future studies of gene expression in transfected keratinocytes.
Lee
J I
JI
Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York, Stony Brook 11794-8702.
Taichman
L B
LB
D001358:Avian Sarcoma Viruses / Q000235:genetics*; D002454:Cell Differentiation; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D000078404:Epidermal Cells; D004817:Epidermis / Q000201:enzymology / Q000502:physiology; D005544:Forecasting; D005786:Gene Expression Regulation; D005796:Genes; D005814:Genes, Viral; D006801:Humans; D007633:Keratins; D012106:Research; D013997:Time Factors; D014162:Transfection
D007633:Keratins; D015500:Chloramphenicol O-Acetyltransferase