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Published on: June 24, 2011
Long-term in vivo gene expression in mouse kidney using φC31 integrase and electroporation
Yuki Otani1, Shigeru Kawakami, Hidefumi Mukai
1Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University , Kyoto , Japan .
Background:
Achieving long-term gene expression in kidney will be beneficial for gene therapy of renal and congenital diseases, genetic studies constructing animal disease models, and the functional analysis of disease-related genes.
Purpose:
The purpose of this study was to develop an in vivo long-term gene expression system in murine kidney using φC31 integrase.
Methods:
Gene expression in cultured RENCA, TCMK-1, and HEK293 cells was assessed. The long-term in vivo gene expression system in the kidney was achieved by co-transfecting 5 µg of pORF-luc/attB as a donor plasmid and 20 µg of pCMV-luc as a helper plasmid into the right kidney of mice by electroporation. Luciferase expression levels were measured to determine longevity of the expression.
Results:
Significantly high luciferase expression levels were observed in cultured RENCA, TCMK-1, and HEK293 cells over 1 month compared with controls (non-integrase system). The luciferase cDNA sequence was integrated at a pseudo attP site termed mpsL1. In vivo luciferase expression levels in the integrase group were sustained and significantly higher than those in the control group over 2 months. Furthermore, φC31 integrase-transfected cells had less genomic DNA damage caused by integrase expression.
Discussion And Conclusion:
These results demonstrated that the φC31 integrase system could produce long-term (2 months) in vivo gene expression in mouse kidney.
