title: A study of the expression of functional human coagulation factor IX in keratinocytes using a nonviral vector regulated by K14 promoter.
abstract: Ex vivo gene therapy requires a suitable bioreactor for production and delivery of the gene products into a target tissue, and keratinocyte is suitable model in this regard because of its potential for systemic release of proteins. To establish a keratinocyte-specific expression system, a mammalian-based expression plasmid equipped with a 2,240-bp fragment from the human keratin 14 (k14) gene enhancer/promoter region was constructed and used for the insertion of the human coagulation factor IX (hFIX)-cDNA downstream the K14-derived regulatory elements. The 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 (rhFIX) was evaluated by performing coagulation test as well as enzyme-linked immunosorbent assay on the cultured media collected from the transfected cells at various stages. The rhFIX corresponding transcript and protein were confirmed by performing reverse transcription PCR as well as immunoblotting experiments, respectively. Based on the coagulation activities obtained from the conditioned media of nine isolated clones, the hFIX expression levels vary from 5% to 39% of normal human plasma. Expression levels of the hFIX obtained in this study are comparable to those reported for viral systems. The obtained data supported the potential of keratinocyte for the expression and secretion of biologically active rhFIX and underscore the importance of the examined cis sequences for enhancing gene expression in a mammalian expression system. Besides, it has provided means for further bioengineering strategies to improve the expression efficiency of the hFIX in keratinocytes and other mammalian host cells.
authors.affiliation: Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Seyed Javad
authors.initials: SJ
authors.lastname: Hosseini
authors.forename: Alireza
authors.initials: A
authors.lastname: Zomorodipour
authors.forename: Razieh
authors.initials: R
authors.lastname: Jalal
authors.forename: Farzaneh
authors.initials: F
authors.lastname: Sabouni
authors.forename: Fariba
authors.initials: F
authors.lastname: Ataei
mesh_terms: D002478:Cells, Cultured; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D053547:Keratin-14 / Q000235:genetics* / Q000378:metabolism; D015603:Keratinocytes / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C507540:KRT14 protein, human; D053547:Keratin-14; D011994:Recombinant Proteins; D005164:Factor IX