EXPRESSION OF THE HUMAN COAGULATION FACTOR IX MINIGENES IN CULTURED HUMAN KIDNEY CELLS

Sam, M.R and Zomorodipour, A.R and Haddad Mashrizeh, A.A and Ghorbani, M and Kardar, G.A (2016) EXPRESSION OF THE HUMAN COAGULATION FACTOR IX MINIGENES IN CULTURED HUMAN KIDNEY CELLS. The Journal of Urmia University of Medical Sciences, Vol. 27(7), October 201, 27 (7). pp. 589-597.

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Abstract

Hemophilia B is caused by either functional deficiency or lack of the human coagulation factor IX (hFIX). The current protein-based therapy with plasma-derived proteins increases, the risk of blood-borne pathogens transmission. Therefore, replacement therapy with recombinant hFIX (rhFIX) is an attractive alternative to plasma derived hFIX concentrates. In order to express and produce rhFIX protein, an efficient expression vector with suitable cis-regulatory elements such as intronic sequences is required. Materials & Methods: Four hFIX-expressing plasmids with or without human β-globin (hBG) introns (intron I and intron II) inside the hFIX-cDNA and the Kozak element were constructed and introduced into the Hek-293T cells using transfection method. Next, the efficacies of the plasmids were evaluated by performing ELISA on cultured media during 3 days of post-transfection as well as semi-quantitative RT-PCR. Results: The highest hFIX expression levels were obtained from the intron-less and intron-I containing plasmids after 3 days of transfection. The first hBG intron was more effective than the second one. Furthermore, the highest mRNA level was obtained from the intron-less construct. Conclusion: Intron-less and intron-I containing plasmids were more effective compared to other constructs for expression of hFIX. Application of the hBG intronic sequences reduced the hFIX expression levels, probably due to improper splicing of the hBG introns from the hFIX pre-mRNAs

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