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

Genetics and Molecular Research : GMR
A M FontesDimas T Covas

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/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...Continue Reading

References

Aug 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·J J TooleR J Kaufman
May 20, 1988·Cell·B Furie, B C Furie
Aug 1, 1972·Proceedings of the National Academy of Sciences of the United States of America·S N CohenL Hsu
Jun 21, 2001·Proceedings of the National Academy of Sciences of the United States of America·T V PestovaC U Hellen
Jan 1, 1993·Biotechnology Advances·F ManningR O'Kennedy
Dec 24, 2003·Seminars in Hematology·Marilyn J Manco-Johnson
May 4, 2004·Methods : a Companion to Methods in Enzymology·Brian DalbyValentina C Ciccarone
Dec 1, 2004·Haemophilia : the Official Journal of the World Federation of Hemophilia·A A AshraniN S Key
Jun 11, 2005·Journal of Thrombosis and Haemostasis : JTH·N BovenschenK Mertens
Nov 28, 2007·Biotechnology Journal·Theo Dingermann
May 2, 2008·Thrombosis and Haemostasis·Steven W Pipe
Jun 25, 2008·Haemophilia : the Official Journal of the World Federation of Hemophilia·S W PipeD Lillicrap
Aug 7, 2008·Molecular Therapy : the Journal of the American Society of Gene Therapy·Alex H ChangMichel Sadelain
May 29, 2009·Haemophilia : the Official Journal of the World Federation of Hemophilia·S W Pipe
Jun 17, 2010·The Journal of Clinical Investigation·Brian C BeardHans-Peter Kiem
Nov 19, 2010·Expert Review of Hematology·Rodney M Camire
Dec 17, 2010·HPB : the Official Journal of the International Hepato Pancreato Biliary Association·Satoshi YokoyamaNigel Heaton
Sep 17, 2011·Journal of Thrombosis and Haemostasis : JTH·S W PipeW H Velander

❮ Previous
Next ❯

Citations

Sep 13, 2015·Oncotarget·Sergiu ChiraIoana Berindan-Neagoe
Apr 7, 2020·Biotechnology and Applied Biochemistry·Eduardo F MufarregeRicardo B Kratje
Oct 19, 2017·International Journal of Oncology·Ricardo Bonfim-SilvaAparecida Maria Fontes

❮ Previous
Next ❯

Related Concepts

Related Feeds

Blood Clotting Disorders

Thrombophilia includes conditions with increased tendency for excessive blood clotting. Blood clotting occurs when the body has insufficient amounts of specialized proteins that make blood clot and stop bleeding. Here is the latest research on blood clotting disorders.