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Title

THE FUNCTION OF INTRON-1 OF HUMAN BETA GLOBIN GENE ON THE EXPRESSION OF BIOLOGICALLY ACTIVE HUMAN FACTOR IX IN A MAMMALIAN CELL LINE

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 Start Page 209 | End Page 223

Abstract

 Background and Objectives: Several studies have shown the positive effects of INTRONS on the expression of heterologous genes in mammalian hosts. In this study, transient expression increment of hFIX in the presence of intron-1 of human beta-glubin was studied.Materials and Methods: The intron-1 of human beta-glubin (hBG) was introduced into the human FACTOR IX cDNA in donor/acceptor site between exons 1 and 2. The constructed hFIX mini-gene and a native hFIX were inserted separately in two expression vectors next to the CMV promoter. After verification, the two recombinant plasmids with and without intron were used to transfect Chinese Hamster Ovary (CHO) cells. The cultured media taken from the tansfected cells were examined for coagulation activity with a single step clotting test performed on FIX-deficient plasma. Then, to confirm the expression of recombinant hFIX by transfected cells, RT-PCR test was conducted.Results: The preliminary data obtained from the expression analysis of the two groups of transfected cells in comparison with the cells with parental plasmid (as negative control) indicate of an increase of about 16 to 62% in coagulation activity of both groups of transfected cells. The same data show an enhanced hFIX coagulation activity of about 1 to 28% in the culture media taken from the cells with intron-containing hFIX-cDNA. Furthermore, RT-PCR confirmed correct splicing process and expression of hFIX from both transfected cells. Conclusions: The positive function of hBG intron 1 on the expression of hFIX in CHO CELLS was shown.Besides, the constructed plasmids have provided tools for analysis of the stability of the transfected cells for production of biologically active hFIX in a systematic approach.

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