High expression of a B-domain deleted factor VIII gene in a human hepatic cell line.
The expression of a modified human coagulation factor VIII cDNA in a human liver-derived cell line is described. A B-domain deleted FVIII (rVIIIdB928) cDNA controlled by a strong viral promoter/enhancer was linked to a dominant selection-/amplification marker and transfected into the human hepatic cell line SK-HEP-1. By means of this system, up to 3.5 U rFVIIIdB928/10(6) cells x 24 h could be detected immediately after selection without gene amplification. This level is orders of magnitude higher than that obtained in Chinese hamster ovary (CHO) f1p4s under the same conditions. Efficient expression of rFVIIIdB928 in SK-HEP-1 cells was temperature dependent, a 4-fold higher level of activity was achieved in culture supernatants at decreased incubation temperatures of 28 degrees C. This system allows the production of high amounts of recombinant rFVIIIdB928 without time and labour consuming gene amplification procedures.
Herlitschka
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Hyland-IMMUNO AG, Biomedical Research Center, Donau, Austria.
Schlokat
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Falkner
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FG
Dorner
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D002460:Cell Line; D005169:Factor VIII / Q000235:genetics / Q000302:isolation & purification*; D005784:Gene Amplification / Q000235:genetics; D006467:Hemophilia A / Q000235:genetics; D006801:Humans; D008099:Liver / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D017384:Sequence Deletion / Q000235:genetics; D013696:Temperature; D013917:Thrombin / Q000378:metabolism; D014162:Transfection / Q000235:genetics
D011994:Recombinant Proteins; D005169:Factor VIII; D013917:Thrombin