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Creation of Tenecteplase-Producing Cho Cell Line Using Site-Specific Integrase From the Phage Φc31



Dormiani K1, 2 ; Khazaie Y1 ; Forouzanfar M1 ; Ghaedi K1, 3 ; Mofid MR4 ; Karbalaie K5 ; Karamali F5 ; Calos MP6 ; Esfahani MHN1
Authors
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Authors Affiliations
  1. 1. Molecular Biotechnology Department, Royan Institute for Animal Biotechnology, ACECR, Isfahan, P.O.Box: 19395-4644, Iran
  2. 2. Pharmaceutical Biotechnology Department, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
  3. 3. Biology Department, School of Science, University of Isfahan, Isfahan, Iran
  4. 4. Biochemistry Department, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
  5. 5. Cell and Molecular Biology Department, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran
  6. 6. Genetics Department, Stanford University, School of Medicine, Stanford, CA, United States

Source: Yakhteh Published:2010

Abstract

Objective: The aim of this study was to produce a stable CHO cell line expressing tenecteplase. Materials and Methods: In the first step, the tenecteplase coding sequence was cloned in a pDB2 vector containing attB recognition sites for the phage φC31 integrase. Then, using lipofection, the CHO cells were co-transfected with constructed recombinant Plasmid encoding tenecteplase and attB recognition sites and the integrase coding sequence containing pCMV-Int plasmid. As the recombinant plasmid contained the neomycin resistance gene (neo), stable cells were then selected using G418 as an antibiotic. Stable transformed cells were assessed using genomic PCR and RT-PCR. Finally, the functionality of tenecteplase was evaluated on the cell culture media. Results: our results indicated that tenecteplase coding sequence was inserted into the CHO cell genome and was successfully expressed. Moreover, tenecteplase activity assessment indicated the presence of our functional tenecteplase in the cell culture medium. Conclusion: Considering the data obtained from this study, φC31 integrase can be used for the production of a stable cell line and it be used to introduce ectopic genes into mammalian cells.
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