Creation of Tenecteplase-Producing CHO Cell Line Using Site-Specific Integrase from the Phage φC31

Author(s):

Kianoush Dormiani , Pharm.D. , Yahya Khazaie , Pharm.D. , Mahboobeh Forouzanfar , Kamran Ghaedi * , Mohammad Reza Mofid , Khadijeh Karbalaie , Fereshteh Karamali , Michele P. Calos , Mohammad Hossain Nasr Esfahani *

Message:
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.
Language:
English
Published:
Cell Journal (Yakhteh), Volume:12 Issue: 2, 2010
Page:
207
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