Functions of the Heterologous Intron-Derived Fragments Intra and Extra Factor IX-cDNA Coding Region on the Human Factor IX Expression in HepG2 and Hek-293T Cells

Iranian Journal of Biotechnology
Mohammad Reza SamSohrab Sam

Abstract

Human FIX (hFIX) gene transfer into hepatocytes has provided a novel approach for treatment of hemophilia B. To obtain an improved expression of hFIX, the functional hFIX-expressing plasmids with appropriate intron-derived fragments which facilitate transcription and promote an efficient 3'-end formation of mRNAs are required. We aim to evaluate the functions of the heterologous intron-derived fragments intra and extra hFIX-cDNA coding region with respect to the hFIX expression in the hepatocytes and kidney cells. HepG2 cells as differentiated hepatocytes and Hek-293T cells as embryonic kidney cells were transfected with the different hFIX-expressing plasmids containing various combinations of the two human beta-globin (hBG) introns within the hFIX-cDNA and Kozak sequence. In the next stage, as a hepatocyte-specific sequence, the rat aldolase B intronic enhancer sequence (rABE), was isolated from the first intron of the rat aldoase B gene and inserted within the upstream CMV promoter (CMVp) and efficacies of the engineered vectors were investigated in the stably-transfected HepG2 cells. Our data indicate that the intron-less construct and hBG intron-I containing construct are more effective with regard to hFIX expression compar...Continue Reading

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Methods Mentioned

BETA
PCR
transfection
ELISA

Software Mentioned

Ensembl
SPSS

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