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[Retroviral vector-mediated HSVtk gene expression and acquisition of high titer recombinant virus]
Zhao-jun Zeng1, Wei-xin Hu, Sai-qun Luo
1Molecular Biology Research Center of Xiangya Medical College, Central South University, Changsha 410078, China.
Objective:
To explore the HSVtk gene expression mediated by the retroviral vector and to obtain high titer recombinant retroviral virus.
Methods:
The recombinant vector pRevTRE/HSVtk was constructed by inserting HSVtk gene into pRevTRE. The recombinant retrovirus, which was produced from cloned PA317 cells screened by hygromycin B after "micro-pingpong" technique transferring with pRevTRE/HSVtk plasmids DNA by using modified calcium phosphate precipitation method. HSVtk gene expression was performed on target cells and virus titers were detected in different cultured temper, time and sodium butyrate concentration.
Results:
The recombinant retroviral vector pRevTRE/HSVtk was constructed and HSVtk gene expression was detected on target cells after they were infected with the recombinant retrovirus.
Conclusion:
High titer of retroviruses could be obtained in the culture medium of PA317 cell line through "micro-pingpong" technique at 30 hours and 10 mmol/L sodium butyrate concentration followed by frozen ultrafiltration.
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