[Non-viral vector mediating human coagulation factor VIII gene expression in mouse 32D cell line].
To investigate the non-viral vector mediating human coagulation factor VIII gene expression in mouse 32D cell line, a recombinant plasmid vector, pRC/RSV-hFVIIIBDcDNA, was constructed by cloning B-domain-deleted (Delta760aa-1639aa) human factor VIII cDNA (hFVIIIBDcDNA) into plasmid vector, pRC/RSV. The plasmid RC/RSV-hFVIIIBDcDNA was then transfected by means of SuperFect Transfection Reagent into mouse 32D cell line. After screening with G418, the procoagulant activity (hFVIII:C) and antigen (hFVIII:Ag) of human factor VIII in the culture medium were detected using one-stage method and ELISA, respectively. Furthermore, RT-PCR was performed to observe the transcription of hFVIIIBDcDNA. The results showed that human coagulation factor VIII protein existed in culture medium with hFVIII:C up to 2.01 U/(10(6) cell x 24 hours) and hFVIII:Ag to 450.08 ng/(10(6) cell x 24 hours). RT-PCR displayed mRNA of hFVIIIBDcDNA in 32D cells. It is concluded that the recombinant plasmid RC/RSV-hFVIIIBDcDNA can successfully express human FVIII in mouse 32D cell line, and hFVIII expressed in vitro presents the similar coagulant activity to the native hFVIII existing in normal human plasma.
Yin
Jun
J
Ruijin Hospital, The Shanghai Second Medical University, Shanghai Institute of Hematology, Shanghai 200025, China.
Wang
Hong-Li
HL
Wang
Xue-Feng
XF
Qu
Bin
B
Wu
Wen-Man
WM
Ding
Qiu-Lan
QL
Fu
Qi-Hua
QH
Qi
Zheng-Wu
ZW
Wang
Zhen-Yi
ZY
D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018076:DNA, Complementary; D005169:Factor VIII