Long-term in vivo gene expression in mouse kidney using ϕC31 integrase and electroporation

Abstract

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.

• ϕC31 integrase
• electroporation
• gene therapy
• in vivo long-term gene expression
• kidney
• plasmid DNA
• transfection

Acknowledgements

The authors are grateful o Dr. T. Kusakabe (Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences) for the ϕC31 integrase expression plasmid, pENTR11-int C-NLS, and pZerofC31attB3xP3EGFP containing an attB site. The authors thank Yoshiaki Umemoto for his skilful technical support in the in vitro experiments.

Declaration of interest

This research was supported in part by a grant-in-aid for Challenging Exploratory Research (26670082, S. K.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by J. F. E. (the Japanese Foundation for Research and Promotion of Endoscopy) Grant (S. K.). The authors alone are responsible for the content and writing of the paper.