Non-viral vector mediating human coagulation factor VIII gene expression in mouse 32D cell line.
- Author:
Jun YIN
1
;
Hong-Li WANG
;
Xue-Feng WANG
;
Bin QU
;
Wen-Man WU
;
Qiu-Lan DING
;
Qi-Hua FU
;
Zheng-Wu QI
;
Zhen-Yi WANG
Author Information
1. Ruijin Hospital, The Shanghai Second Medical University, Shanghai Institute of Hematology, Shanghai 200025, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cell Line;
DNA, Complementary;
genetics;
Enzyme-Linked Immunosorbent Assay;
Factor VIII;
genetics;
metabolism;
Gene Expression Regulation;
Humans;
Mice;
Plasmids;
genetics;
Reverse Transcriptase Polymerase Chain Reaction;
Transfection
- From:
Journal of Experimental Hematology
2004;12(6):721-725
- CountryChina
- Language:Chinese
-
Abstract:
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.
