Non-viral vector mediating human coagulation factor VIII gene expression in mouse 32D cell line / 中国实验血液学杂志
Journal of Experimental Hematology
; (6): 721-725, 2004.
Article
em Zh
| WPRIM
| ID: wpr-347876
Biblioteca responsável:
WPRO
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 (hFVIIIC) and antigen (hFVIIIAg) 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 hFVIIIC up to 2.01 U/(10(6) cell x 24 hours) and hFVIIIAg 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.
Texto completo:
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Índice:
WPRIM
Assunto principal:
Plasmídeos
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Fator VIII
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Ensaio de Imunoadsorção Enzimática
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Transfecção
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Linhagem Celular
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Regulação da Expressão Gênica
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DNA Complementar
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Reação em Cadeia da Polimerase Via Transcriptase Reversa
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Genética
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Metabolismo
Limite:
Animals
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Humans
Idioma:
Zh
Revista:
Journal of Experimental Hematology
Ano de publicação:
2004
Tipo de documento:
Article