Abstract

  Background and Objectives

  Keratinocyte is a suitable model for ex vivo gene expression strategies for systemic release of a protein. In this regard, keratincyte-specific regulatory elements are especially attractive candidates for keratinocyte–mediated gene expression. In the present study, a vector was designed for specific expression of recombinant protein in human epidermal keratinocytes using human keratin 14 gene promoter. The potential of the constructed plasmid was shown by the expression of human FIX-cDNA in human primary keratinocytes.

  Materials and Methods

  A fragment containing human keratin14 gene promoter was inserted in the pcDNA3 plasmid. The human FIX cDNA isolated from a human liver cDNA library was inserted into the recombinant plasmid (phPK 14 H). Human epidermal keratinocytes isolated from neonatal foreskin were cultivated in keratinocyte serum-free medium and transfected with the newly made recombinant plasmid (pK14hFIX) using fugene-6. About 72 hours after transfection, cultured media were collected for expression analysis, and the cells were transferred into selective media containing geneticin. Incubation continued until the appearance of 9 colonies. The expression of human FIX-cDNA was examined by performing one-stage clotting assay as well as RT-PCR.

  Results

  The clotting times obtained from the cultured media of different transfection lines were compared to of the negative controls. Indeed the coagulation activity of the expressed rhFIX in the cultured media was detectable both before and after geniticin selection of the transfected keratinocytes. The expression of hFIX-cDNA in the transfected cells was also confirmed by performing RT-PCR.

  Conclusion

  Our preliminary results support the idea that human keratinocyte has potential for the production and secretion of biologically active FIX. The expression plasmid constructed in this study, has provided useful means for the expression of different proteins of medical importance in keratinocyte for further biochemical and cellular studies.

  Key words: Factor IX ، Keratinocyte, Keratin-14, Somatic gene therapy