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We developed a tissue suction-mediated transfection method (suction method) as a relatively reliable and less invasive technique for in vivo transfection. In this study, we determined hepatic transgene expression characteristics in the mouse liver, using a suction device, collecting information relevant to gene therapy and gene functional analysis by the liver suction method. To achieve high transgene expression levels, we developed a suction device with four holes (multiple hole device) and applied it to the larger portion of the left lateral lobe of the mouse liver. Hepatic transfection with physical stimuli was potentially controlled by activator protein-1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We examined the spatial distribution of transgene expression in the suctioned lobe by 2-dimensional imaging with histochemical staining and 3-dimensional multicolor deep imaging with tissue clearing methods. Through monitoring spatial distribution of transgene expression, the liver suction method was used to efficiently transfect extravascular hepatocytes in the suction-deformable upper lobe of the liver. Moreover, long-term transgene expression, at least 14 d, was achieved with the liver suction method when cytosine-phosphate-guanine (CpG)-free plasmid DNA was applied.

PhiC31 integrase has the potential to achieve long-term transgene expression because of site-specific and unidirectional recombination. In this study, plasmid DNA (pDNA) encoding Gaussia luciferase (Gluc) cDNA was constructed and the optimal condition for long-term gene expression using phiC31 integrase in mouse liver after hydrodynamic injection was investigated. Gluc is secreted and thus allows quantification of its expression level in blood and easy determination of the expression time-course. Mice were co-transfected with 25 µg donor pDNA (pORF-Gluc/attB) and different amounts of helper pDNA (pCMV-int; from 1 to 50 µg). Serum Gluc expression was evaluated during 120 d. The most highly sustained gene expression was obtained when 10 µg of helper pDNA was co-transfected in ICR and C57BL/6 mice. However, the Gluc expression in C57BL/6 mice was slightly lower and less durable than that in ICR mice. Little hepatic damage caused by phiC31 integrase was observed during 120 d in ICR and C57BL/6 mice.