[A preliminary study on the in vitro eukaryotic expression of human factor VIII cDNA].
OBJECTIVE
To evaluate the expression efficiency of a cDNA sequence of human clotting factor VIII (4.7 kb, B domain-deleted) in in vitro systems.


METHODS
After insertion of the cDNA into several mammalian expression vectors, such as retroviral vector pMSCV, EB virus-based vector pGRE5.2/EBV and eukaryotic expression plasmid pCI, the expression of these constructs were tested in a variety of cells.


RESULTS
All the three kinds of constructs-pCI-VIII, pGRE5.2/EBV-VIII and pMSCV-VIII were able to direct FVIII synthesis in NIH3T3, Hela and Bosc23 cells, respectively, while the pMSCV-VIII and pGRE5.2/EBVVIII produced relatively high levels of FVIII activity (up to 0.7 units/ml and 2.0 units/ml from 24 h to 48 h, respectively, after transfection with lipofectamine). The three forms of pMSCV-VIII vector worked in a similar efficacy in Bosc 23 cells, but this function was not detected in NIH3T3, psi-Crip and GP + E86 as well as 32DC13 cells in a transient transfection assay. Moreover, the NIH3T3 and 32DC13 cells infected with culture supernatant from pMSCV-VIII transfected-Bosc23 cells (as packaging cells) unexpectedly did not produce detectable FVIII activity.


CONCLUSIONS
Apart from the design and construction of vectors, target cell selection may play a crucial role in the efficient expression of the FVIII cDNA.
Zhu
J
J
Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025.
Wang
H
H
Yu
L
L
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D005169:Factor VIII / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D051379:Mice
D005169:Factor VIII; D004247:DNA