Yoshinori Masaki

Gene transfer into mammalian cells by use of a nanosecond pulsed laser-induced stress wave

Plasmid DNA has been successfully delivered to mammalian cells by applying a nanosecond pulsed laser-induced stress wave (LISW). Cells exposed to a LISW were selectively transfected with plasmids coding for green fluorescent protein. It was also shown that transient, mild cellular heating (approximately 43 degrees C) was effective in improving the transfection efficiency.

In vitro gene transfer to mammalian cells by the use of laser-induced stress waves: Effects of stress wave parameters, ambient temperature, and cell type

Laser-mediated gene transfection has received much attention as a new method for targeted gene therapy because of the high spatial controllability of laser energy. We previously demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISWs). In the present study, we investigated the dependence of transfection efficiency on the laser irradiation conditions and hence stress wave conditions in vitro. We measured characteristics of LISWs used for gene transfection. For NIH 3T3 cells, transfection efficiency was evaluated as functions of laser fluence and number of pulses. The effect of ambient temperature was also investigated, and it was found that change in ambient temperature in a specific range resulted in drastic change in transfection efficiency for NIH 3T3 cells. Gene transfection of different types of cell lines were also demonstrated, where cellular heating increased transfection efficiency for nonmalignant cells, while heating decreased transfection efficiency for malignant cells.

Experience with individual receipt confirmation system and the university primary mail service

Before experiencing last years H1N1 pandemic, we had happened to realize the need for a reliable communication method to provide all staff members and students with information about disease and emergency situations. Our legacy mail system was good for sending information, but it was difficult for the sender to get receipt confirmation. In July 2009, we introduced a new mail delivery system providing an official mail address to all the staff members in our university. In addition, we implemented a new system to send e-mails to all staff members and students and to receive the confirmation that the e-mail had been read. Now it is possible for us to send e-mail according to a specified campus or position code such as professor, part time staff, and students. The e-mail recipients are expected to respond to it by clicking the URL in the message text. The system records the user responses by the ID number, event code, time, etc. into the database. Students are expected to answer the questionnaire followed by the confirmation page. The questionnaire asks the whether a student has become infected with the influenza, about recovery, if they belong to clubs, their class schedule, etc. if so. It makes it possible for us to grasp the individuals situation and to get the information to contact them.

Staining of Living Merkel Cells with FM Dyes

Live Merkel cells are known to incorporate a fluorescent dye, quinacrine (3,3′,4′,5,7-pentahydroxyflavone). Quinacrine fluorescence in the cells is, however, quickly lost and quinacrine-stained Merkel cells become difficult to identify in tissue culture. To find dyes that stay in the cells for a long period of time, we tested many fluorescent dyes and found that FMI-43 (N-(3triethylammoniumpropyl)-4-(4-(dibutylamino)-styryl)pyridinium dibromide) is a useful marker for live Merkel cells. In the rat footpad skin, FM1-43 was revealed to stain most of the live Merkel cells that were already stained with quinacrine. Merkel cells in sinus hair follicles were also stained with FM1-43 dye. The fluorescence intensity of the FM dye was stronger than that of quinacrine, and the shape of the cells was more distinct in the FMI-43-stained cells. We thus conclude that the FM dye is a powerful tool for tracing live Merkel cells in in vitro experiments. Moreover, the finding that Merkel cells incorporate the FM dye suggests that vesicles in Merkel cells are likely to represent recycling in a manner similar to those in neurons and secretory cells.

In vitro gene transfer by the use of laser-induced stress wave: Effect of laser irradiation conditions on transfection efficiency

Laser-mediated gene transfection is very attractive as a new method for targeted gene therapy because of its high spatial controllability of laser energy. Previously, we demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISW). In this study, we investigated the experimental conditions to increase transfection efficiency in vitro. By applying single-pulse LISW, transfection efficiency was increased with increasing laser fluence. Increase in the number of laser pulses increased transfection efficiency for laser fluences up to 1.3 J/cm2, but at higher fluences (>1.7 J/cm2), efficiency showed saturation tendency. Temperature dependence of transfection efficiency was also investigated.

In vitro gene transfer by the application of laser-induced stress wave: effect of cellular heating

We previously demonstrated in vitro that the simultaneous application of cellular heating and a laser-induced stress wave (LISW) enhanced the uptake of porfimer sodium (Photofrin) by cells. In this study, we attempted to apply this technique to gene transfer to cultured cells. LISW and/or a transient mild (~43°C) heating been applied to deliver plasmid coding for green fluorescent protein (EGFP) to NIH-3T3 cells. It was found that simultaneous application of an LISW and the heating significantly increased the transfection efficiency by a factor of 2.5 when compared with that for the cells treated with an LISW alone.