Non-invasive visualization of in vivo electroporation-mediated gene expression in living mouse

Effective gene transfer and long term expression of the transgene, and its monitoring is important for optimized gene therapy and study of gene expression in vivo. In vivo electroporation is receiving much attention as a mean to increase the efficacy of non-viral gene and drug delivery into tumor and target tissues. Optical imaging is an attractive way to non-invasively evaluate the expression of a transferred gene. In this study, (1) we established the subcutaneous tumor model in mice by inoculation of transformed human embryonic kidney cell line (HEK293), (2) we performed the electroporation-assisted transfer of a plasmid DNA encoding the DsRed gene constitutively driven by composite CAG promoter to xenografted tumor, and (3) we monitored with IVIS® lumina optical imaging system (Xenogen) to confirm the transfection procedure and to determine whether expression levels were maintained for the duration of the experiment. The plasmid DNA was injected directly into tumor, followed by electroporation with CUY21SC electoroporator (NEPA Gene). In vivo expression level was evaluated by monitoring the real time spatial distribution and time dependence of DsRed fluorescence light emission. The emission of light was quantified as photons per second using Living Image® software version 2.6 (Xenogen). The expression of DsRed in tumor model was detected on day 1 and gradually increased up to the maximal level on day 6 or 7. DsRed fluorescence light emission was also detected in ex vivo optical imaging of the tumor and confocal microscopy of tumor cryosection confirming the in vivo findings. The imaging experiments as a whole demonstrated that detectable levels of DsRed are observed only at the site of plasmid injection and not at distant sites, and that plasmid persists long-term in electroporatively-transfected cells in vivo without the CAG promoter silencing by the procedure. In conclusion, optical imaging is a useful and suitable for monitoring the spatial and temporal efficacy of electroporation methods for gene transfection in an animal tumor model.第２回日本分子イメージング学会総会・学術集