| Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector. | |
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MedLine Citation:
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PMID: 22576836 Owner: NLM Status: In-Data-Review |
Abstract/OtherAbstract:
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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. |
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Authors:
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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:
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Type: Journal Article Date: 2012-03-22 |
Journal Detail:
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Title: Genetics and molecular research : GMR Volume: 11 ISSN: 1676-5680 ISO Abbreviation: Genet. Mol. Res. Publication Date: 2012 |
Date Detail:
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Created Date: 2012-05-11 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 101169387 Medline TA: Genet Mol Res Country: Brazil |
Other Details:
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Languages: eng Pagination: 775-89 Citation Subset: IM |
Affiliation:
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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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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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