We have designed a keratinocyte-specific expression plasmid equipped with the K14-promoter by inserting a 2240bp BglII/HindIII fragment, containing the upstream regulatory elements of the K14 gene, in a digested pcDNA3 plasmid, from which the CMV promoter had been deleted. Subsequently human epidermal keratinocyte, isolated from neonatal foreskin, was transfected with the recombinant plasmid, based on liposome mediated transfection technique. The transfection step was continued for either 6 or 21 hours. The transfected cells were then subjected for the expression analysis. The K14-promoter-deriven expression of rhFIX in the transfected cells was examined by performing one-stage clotting assay, using hFIX immuno-depleted plasma on the cultured media on the day 3 of post-transfection. The pro-coagulation activity of the cultured media collected from the transfected keratinocytes indicates in the expression of hFIX by the transfected cells. The expression of recombinant hFIX in the transfected cells was also confirmed by RT-PCR.

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