We report here development of a novel gene trap method in zebrafish using the Tol2 transposon system. First, we established a highly efficient transgenesis method in which a plasmid DNA containing the Tol2 transposon vector and the transposase mRNA synthesized in vitro were coinjected into one-cell stage embryos. The transposon vector inserted in the genome could be transmitted to the F1 progeny at high frequencies, and regulated gene expression by a specific promoter could be recapitulated in transgenic fish. Then we constructed a transposon-based gene trap vector containing a splice acceptor and the GFP gene, performed a pilot screen for gene trapping, and obtained fish expressing GFP in temporally and spatially restricted patterns. We confirmed the endogenous transcripts were indeed trapped by the insertions, and the insertion could interfere with expression of the trapped gene. We propose our gene trap approach should facilitate studies of vertebrate development and organogenesis.

The Tol2 element of the medaka fish Oryzias latipes belongs to the hAT family of transposons (hobo͞Ac͞Tam3). We report here identification of a functional transposase of Tol2 that is capable of catalyzing its transposition in the germ line of zebrafish Danio rerio. A transcript produced from Tol2 encodes a putative transposase. Zebrafish fertilized eggs were coinjected with mRNA transcribed in vitro, using cDNA of the Tol2 transcript as a template and a plasmid DNA harboring a mutant Tol2, which had a deletion in the putative transposase gene but retained necessary cis sequences. The injected fish were raised to adulthood and mated to noninjected fish, and genomic DNA of the progeny fish were analyzed by PCR and Southern hybridization. Half of F 1 fish obtained from one of eight injected fish contained the Tol2 DNA in their genomes but not the vector portion. Among these F 1 fish, Tol2 insertions at four different loci were identified, and some F1 fish carried two or three different Tol2 insertions, indicating that the germ line of the founder fish is highly mosaic. Sequencing analyses revealed that, in all cases, Tol2 was surrounded by zebrafish genomic sequences, and an 8-bp duplication was created at the target site, indicating that Tol2 was integrated in the zebrafish genome through transposition. This study identifies an autonomous member of a DNA-based transposable element from a vertebrate genome. The Tol2 transposon system should thus be used to develop novel transgenesis and insertional mutagenesis methods in zebrafish and possibly in other fishes.

A centre-solenoid-free merging start-up scheme for spherical tokamak plasmas was developed in a University of Tokyo spherical tokamak (UTST) experiment by using outer poloidal field coils. Torus breakdown was initiated at null points and two spherical tokamak plasmas with a total current up to 80 kA were generated inductively. Their merging process provided substantial ion and electron heating by magnetic reconnection. The obtained dependence of heating on plasma current suggests that high-temperature and high-current plasma suitable for neutral beam injection is attainable under the realistic conditions in the merging start-up method.

Floating potential profile was measured around the X-point during high guide field reconnection in UTST merging experiment where the ratio of guide field (Bg) to reconnecting magnetic field (Brec) is Bg/Brec>10. Floating potential measurement revealed that a quadrupole structure of electric potential is formed around the X-point during the fast reconnection phase due to the polarization by inductive electric field. Also, our floating potential measurement revealed the existence of parallel electric field in the vicinity of the X-point. While field-aligned components of inductive electric field (E∥ind) and electrostatic electric field (E∥es) cancel out with each other away from the X-point, E∥ind exceeds E∥es around the X-point, indicating the deviation from ideal MHD criterion within the region. The diffusion region extends in the outflow region and the scale length of region is an order of ion skin depth, which is quite different from the VTF experiment result. Based on the measured magnetic field and electric field profile, our particle trajectory analysis indicates that fast electrons with energies over 300 eV are produced within 1 μs around the X-point in the non-ideal MHD region. These results indicate that production of fast electrons or electron heating are expected to be observed in the vicinity of the X-point.

The lethal effects of gallium citrate in combination with heat were studied using four cell lines, L5178Y, FM3A, P388 and HeLa. Cells were incubated with different concentrations (0.2 2 mM) of gallium citrate at 37 degrees C for 24 h and heated at a range of temperatures from 40-44 degrees C for various time periods up to 6 h in the absence of gallium citrate. Survival and cell viability were determined by clonogenic assay and the dye-exclusion test, respectively. All of the cell lines tested were insensitive to heat below 41 degrees C, but were very sensitive to heat above 43 degrees C. Gallium citrate was cytotoxic to these cell lines at different levels: P388 and HeLa were far more sensitive than L5178Y and FM3A. The killing effects of heat at 41 degrees C were greatly enhanced by gallium citrate in L5178Y and P388 cells. The Arrhenius analysis for the lethal effect of heat, determined by clonogenic assay, in L5178Y cells showed that the transition temperature was remarkably decreased for the gallium-treated cells from approximately 43 degrees C to 41 degrees C. The mechanism for this decrease in the transition temperature may be attributable to the additional effects of gallium citrate on energy metabolism. Preincubation with 0.05 mM gallium citrate at 37 degrees C for 7 days also enhanced heat sensitization at 41 degrees C in L5178Y. This preincubation condition may correspond to the condition for the continuous infusion of gallium that is clinically used for cancer treatment. In contrast, treatment with gallium did not greatly enhance the sensitivity of FM3A or HeLa cells to heat at 41 degrees C, but the effects of gallium were significant.

The killing effects of heat were studied on cultured mammalian cells (L5178Y) pre‐incubated with gallium (Ga) citrate, which is a popular tumor‐imaging diagnostic agent. The cells showed higher sensitivity to heat when they were pre‐incubated with Ga‐citrate. The pre‐incubated cells showed decreased ATP levels, and this may be responsible for the heat‐sensitizing effect.