An enhancer/promoter combination strengthens the expression of blood-coagulation factor VIII in non-viral expression vectors.
We explored the potential of fusion of hepatic locus control region 1 (HCR-1) with HCR-2 to express B-domain-deleted human factor VIII (FVIII) in four cell lines. B-domain-deleted human FVIII expression was controlled by HCR-1/HCR-2, followed by liver specific and ubiquitous promoters. Chimera enhancer HCR-1/HCR-2, followed by cytomegalovirus (CMV) promoter, gave 2-fold more FVIII expression in all cell lines (105.6 +/- 2.8 for Hek-293, 68.8 +/- 3.8 for HepG2, 34.8 +/- 1.3 for CHO, and 27.2 +/- 1.6 ng x mL(-1) x 10(6) cells(-1) for L.N.) when compared to the vector with CMV alone (54.8 +/- 3.3 for Hek-293, 32.4 +/- 1.2 for HepG2, 18.6 +/- 1.1 for CHO, and 10.1 +/- 1.7 ng x mL(-1) x 10(6) cells(-1) for L.N.). Elongation factor 1-alpha gene and human CMV promoters were more efficient than the promoters from the human alpha-1-antitrypsin gene, and fviii was less efficient in hepatic cell lines. HCR-1/HCR-2, followed by strong promoters, increases FVIII expression in vitro. Our results underscore the importance of cis sequences for enhancing in vitro FVIII expression; this may be helpful for designing new strategies to improve heterologous expression systems.
Picanço-Castro
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Hemocentro de Ribeirão Preto, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brasil. v.picanco@hemocentro.fmrp.usp.br
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D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D003587:Cytomegalovirus / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005169:Factor VIII