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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
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]
Total Authors: 7
Affiliation:
[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
Total Affiliations: 5
Document type: Journal article
Source: Genetics and Molecular Research; v. 11, n. 1, p. 775-789, 2012.
Web of Science Citations: 2
Abstract

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)

FAPESP's process: 98/14247-6 - Center for Research on Cell-Based Therapy
Grantee:Marco Antonio Zago
Support type: Research Grants - Research, Innovation and Dissemination Centers - RIDC
FAPESP's process: 08/57877-3 - National Institute of Science and Technology in Cell Therapy
Grantee:Roberto Passetto Falcão
Support type: Research Projects - Thematic Grants