Otani Yuki, Kawakami Shigeru, Mukai Hidefumi, Fuchigami Yuki, Yamashita Fumiyoshi, Hashida Mitsuru
Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University , Kyoto , Japan .
J Drug Target. 2015 Jun;23(5):427-35. doi: 10.3109/1061186X.2014.1002788. Epub 2015 Feb 12.
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.
The purpose of this study was to develop an in vivo long-term gene expression system in murine kidney using φC31 integrase.
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.
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.
These results demonstrated that the φC31 integrase system could produce long-term (2 months) in vivo gene expression in mouse kidney.
在肾脏中实现长期基因表达将有利于肾脏疾病和先天性疾病的基因治疗、构建动物疾病模型的遗传学研究以及疾病相关基因的功能分析。
本研究的目的是利用φC31整合酶在小鼠肾脏中开发一种体内长期基因表达系统。
评估培养的RENCA、TCMK-1和HEK293细胞中的基因表达。通过电穿孔将5μg pORF-luc/attB作为供体质粒和20μg pCMV-luc作为辅助质粒共转染到小鼠右肾中，从而实现肾脏中的长期体内基因表达系统。测量荧光素酶表达水平以确定表达的持续时间。
与对照组（非整合酶系统）相比，在培养的RENCA、TCMK-1和HEK293细胞中，1个月以上观察到显著高的荧光素酶表达水平。荧光素酶cDNA序列整合在一个称为mpsL1的假attP位点。在2个月的时间里，整合酶组的体内荧光素酶表达水平持续且显著高于对照组。此外，φC31整合酶转染的细胞因整合酶表达而导致的基因组DNA损伤较少。
这些结果表明，φC31整合酶系统可在小鼠肾脏中产生长期（2个月）的体内基因表达。
