Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector

Autor(es)

Fontes, A. M. ^{[1, 2, 3]} ; Melo, F. U. F. ^[2] ; Greene, L. J. ^[2] ; Faca, V. M. ^[2] ; Lin, Y. ^{[4, 5]} ; Gerson, S. L. ^{[4, 5]} ; Covas, D. T. ^{[1, 2, 3]}

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Dept Genet, Fac Med Ribeirao Preto, BR-14049 Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Hemoctr Ribeirao Preto, Inst Nacl Ciencia & Tecnol Celulas Tronco & Terap, Hosp Clin, Fac Med Ribeirao Preto, BR-14049 Ribeirao Preto, SP - Brazil

^[3] Univ Sao Paulo, Dept Clin Med, Fac Med Ribeirao Preto, BR-14049 Ribeirao Preto, SP - Brazil

^[4] Case Western Reserve Univ, Case Comprehens Canc Ctr, Cleveland, OH 44106 - USA

^[5] Univ Hosp Case Med Ctr, Seidman Canc Ctr, Cleveland, OH - USA

• Tipo de documento: Artigo Científico
• Fonte: Genetics and Molecular Research; v. 11, n. 1, p. 775-789, 2012.
• Citações Web of Science: 2

Resumo

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/mu g 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. (AU)

• Processo FAPESP: 08/57877-3 - Instituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular - INCTC
• Beneficiário: Roberto Passetto Falcão
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 98/14247-6 - Center for Research on Cell-Based Therapy
• Beneficiário: Marco Antonio Zago
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs