High efficiency production of germ-line transgenic Japanese medaka (Oryzias latipes) by electroporation with direct current-shifted radio frequency pulses.

Although there have been several studies showing the production of transgenic fish through electroporation techniques, success rates have been low and few studies show germ-line integration and expression. When electroporation has been successful, the device used is no longer commercially available. The goal of this experiment was to find an alternative efficient method of generating transgenic Japanese medaka (Oryzias latipes) using a commercially available electroporation device. The Gene Pulser II and RF module (Bio-Rad Laboratories, USA), along with two reporter gene constructs, were used. In contrast to other electroporation devices, which are based on a single pulse with exponential decay or square wave technology, the Gene Pulser II incorporates a direct current (DC)-shifted radio frequency (RF) signal. With this technique, over 1000 embryos can be electroporated in less than 30 min. The plasmid pCMV-SPORT-beta-gal (Invitrogen, USA) was used in the supercoiled form to optimize parameters for gene transfer into single-celled embryos, and resulted in up to 100% somatic gene transfer. Similar conditions were used to generate fish transgenic for both the pCMV-EGFP plasmid (Clontech, USA) and a cytomegalovirus (CMV) driven phytase-EGFP construct. The conditions used were a voltage of 25 V, a percent modulation of 100%, a radio frequency of 35 kHz, a burst duration of 10 ms, 3 bursts, and a burst interval of 1.0 s. Seventy percent of the embryos electroporated with the pCMV-EGFP construct survived to sexual maturity, and of those, 85% were capable of passing the transgene on to their offspring. Transgenic second generation back-crossed (BC2) fry were subjected to Southern blot analysis, which confirmed germ-line integration, and observation for green fluorescence protein, which confirmed protein expression. DC-shifted RF pulses are effective and efficient in the production of transgenic medaka, and germ-line integration and expression can be achieved without linearization of the transgene vector.