Document Detail


Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector.
MedLine Citation:
PMID:  22576836     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1- kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/μg protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.
Authors:
A M Fontes; F U F Melo; L J Greene; V M Faça; Y Lin; S L Gerson; D T Covas
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Publication Detail:
Type:  Journal Article     Date:  2012-03-22
Journal Detail:
Title:  Genetics and molecular research : GMR     Volume:  11     ISSN:  1676-5680     ISO Abbreviation:  Genet. Mol. Res.     Publication Date:  2012  
Date Detail:
Created Date:  2012-05-11     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101169387     Medline TA:  Genet Mol Res     Country:  Brazil    
Other Details:
Languages:  eng     Pagination:  775-89     Citation Subset:  IM    
Affiliation:
Hemocentro de Ribeirão Preto, Instituto Nacional de Ciência e Tecnologia em Células Tronco e Terapia Celular, Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil fontesam@hemocentro.fmrp.usp.br.
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