Key Physiological Phenomena Governing Transgene Expression Based on Tissue Pressure-Mediated Transfection in Mice

Abstract

It is generally recognized that in vivo gene transfection is one of the most important techniques used in the post-genome era. Above all, naked plasmid DNA transfection has attracted much attention because of its advantages including convenience of preparation and handling and lack of toxicity associated with the transfection agents. We have investigated tissue pressure-mediated transfection performed by light and controlled pressure of the target tissue after normal intravenous injection of plasmid DNA. So far, we have demonstrated that plasmid DNA and small-interfering RNA (siRNA) are very efficiently transfected into murine kidney, liver and spleen without causing marked tissue damage. In this study, in order to understand the key physiological phenomena affecting transgene expression, we performed a set of experiments involving tissue pressure-mediated transfection, including the biodistribution and cellular transport of plasmid DNA and activation of transcriptional factors and obtained the following results: i) plasmid DNA transfer to the target tissue and its cells increased although the transferred fraction was small compared to the total administered plasmid DNA, ii) a transient increase in cellular translocation of plasmid DNA was induced, and iii) transcriptional factors were activated. Taking all these results into consideration, it would appear that tissue pressure-mediated transfection enhances plasmid DNA transfer to the target tissue and its cells and also activation of the transcriptional process. This information will allow a better understanding of in vivo transgene expression based on naked plasmid DNA transfection involving tissue pressure-mediated transfection.

Characterization of transgene expression and pDNA distribution of the suctioned kidney in mice

“…Among them, rapid and transient activation of AP-1 and NF-jB have reported in response to a wide range of stimuli (Fujioka et al, 2004;Sen & Smale, 2010). In a previous study, we demonstrated the involvement of NF-jB and AP-1 in efficient transgene expression in vivo using the tissue pressure method (Mukai et al, 2010). Furthermore, Lam et al have reported that mechanical stress, including cyclic stretching, alters the activation and/or nuclear localization of key transcription factors including NF-jB and the c-fos and c-jun subunits of AP-1 in vitro (Lam et al, 2008).…”

“…those of the tissue pressure method (Mukai et al, 2010). Moreover, we visualized nuclear transportation of fluorescent-labeled pDNA by tissue clearing.…”

“…Total mRNA was extracted from the right kidney using an RNeasy V R Mini Kit (QIAGEN, GmbH, Hilden, Germany). The cycling conditions were as follows: initial denaturation at 95 C for 30 s, followed by 50 cycles at 95 C for 5 s and 60 C for 30 s. Gene-specific fluorescence was measured at 60 C. The primers for c-fos, cjun, and GAPDH were the same as those in a previous report (Mukai et al, 2010). Data are shown as relative mRNA levels to GAPDH mRNA levels.…”

Liver Suction-Mediated Transfection in Mice Using a Pressure-Controlled Computer System

“…[8][9][10] In this method, the target organs of the mice are pressed directly after intravenous injection (i.v.) of the naked nucleic acids.…”

Combination of Nanoparticles with Physical Stimuli toward Cancer Therapy

“…1 (iii)) was involved in enhanced transgene expression by hydrodynamics-based transfection, 49,50) ultrasound-mediated transfection, 51) and pressure-mediated transfection. 52) Thus, it is necessary to consider the effect of the physical stimuli on cellular functions in view of future clinical use.…”