Keratinocytes are suitable models for ex vivo gene expression strategies for the systemic release of a protein. To establish a keratinocyte-specific expression system for studying the keratinocyte–mediated expression of a trans-gene product a human factor IX (hFIX) expressing plasmid was constructed by inserting the hFIX-cDNA downstream to a 2200bp keratin 14 (k14) gene promoter in a pcDNA3-based plasmid. Human epidermal keratinocytes isolated from neonatal foreskin were cultivated in keratinocyte serum-free media and transfected with the recombinant plasmid. The K14-promoter-driven expression of recombinant hFIX was evaluated by performing coagulation test as well as enzyme-linked immunosorbent assay on the cultured media collected from the transfected cells. The rhFIX expression was also confirmed by performing RT-PCR, dot and Western blotting. The results support the potential of keratinocytes for the expression of biologically active rhFIX. The highest value of FIX Ag measured by ELISA (as displayed by 106 cells after 24 hours) was estimated to be 180 ng/ml, which is still comparable to those obtained by other groups who used regulatory elements from CMV (250 ng/ml), retrovirus (830 ng/ml) or moloney murine leukemia virus (600 ng/ml) for the expression of hFIX . The current keratinocyte-based expression system has provided means for further bioengineering strategies to improve the expression efficiency of hFIX in keratinocytes.

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