One of the methods to generate transgenic animals is called sperm-mediated gene transfer (SMGT). Mature sperm cells can take up exogenous DNA molecules intrinsically and transfer them into the oocyte during fertilization. This study assessed the effect of dimethyl sulfoxide (DMSO) and electrolyte-free medium (EFM) on DNA uptake (EGFP–N1plasmid) in mouse sperm. Sperms cells cultured in human tubular fluid (HTF) without any treatment were considered as the control group. Sperms cells that were incubated in EFM and HTF with DNA/DMSO at 4°C were classified into EFM and HTF groups. Sperm motility and viability were assessed following treatment. In vitro fertilization (IVF) with sperm in all groups was performed. Fertilization, embryo development and GFP-positive blastocyst rates were analyzed and compared. The result showed that sperm motility and viability in EFM were better than those in the HTF group. The rate of development to reach the blastocyst stage and GFP-positive blastocysts was significantly higher in the EFM group compared with the HTF group (P<0.05). Our data demonstrate that sperm stored in the EFM group can improve the efficiency of SMGT for the generation of GFP-positive blastocysts.

Bright green auto-fluorescence was observed in the umbrellar margin, umbrellar marginal warts, tentacular bulbs, tentacles, and manubrium of laboratory-reared immature (1–14 days old) medusae of Eutima japonica from Japan and China. In vivo microscopic fluorescence spectra showed that the green fluorescence was similar to that of green fluorescent protein (GFP) found in Aequorea victoria, although the maximum emission wavelength (503 nm) was slightly bluer. No fluorescence was detected in the cirri, statocysts, radial canals, velum, or subumbrella of the medusae. The fluorescence distribution pattern in E. japonica more closely resembles that of Eugymnanthea inquilina from the Mediterranean Sea than that of Japanese Eugymnanthea japonica, which is the derived species of E. japonica. This suggests that the common fluorescence pattern is convergently evolved in the former two species, perhaps owing to the as yet unclarified physiological and/or ecological function of GFP and/or GFP-like proteins.

Light-emitting diodes (LEDs) can be easily and inexpensively integrated into modern light microscopes. There are numerous advantages of LEDs as illumination sources; most notably, they provide brightness and spectral control. We demonstrate that for transmitted light imaging, an LED can replace the traditional tungsten filament bulb while offering longer life; no color temperature change with intensity change; reduced emission in the infrared region, which is important for live cell imaging; and reduced cost of ownership. We show a direct substitution of the typical tungsten bulb with a commercially available LED and demonstrated the color stability by imaging a histology section over a wide range of light intensities. For fluorescent imaging, where the typical illumination sources are mercury or xenon lamps, we demonstrate that LEDs offer advantages of providing a longer lifespan, having a more constant intensity output over time, more homogeneous illumination, and significantly lower photon dose. Our LED equipped system was used to image and deconvolve dual fluorescently labeled cells, as well as image cells undergoing mitosis expressing green fluorescent protein–histone 2B complex. The timing of the stages of mitosis is well established as an indicator of cell viability.

Transgenes from transgenic oilseed rape, Brassica napus (AACC genome), can introgress into populations of wild B. rapa (AA genome), but little is known about the long-term persistence of transgenes from different transformation events. For example, transgenes that are located on the crop’s C chromosomes may be lost during the process of introgression. We investigated the genetic behavior of transgenes in backcross generations of wild B. rapa after nine GFP (green fluorescent protein)-Bt (Bacillus thuringiensis) B. napus lines, named GT lines, were hybridized with three wild B. rapa accessions, respectively. Each backcross generation involved crosses between hemizygous GT plants and non-GT B. rapa pollen recipients. In some cases, sample sizes were too small to allow the detection of major deviations from Mendelian segregation ratios, but the segregation of GT:non-GT was consistent with an expected ratio of 1:1 in all crosses in the BC1 generation. Starting with the BC2 generation, significantly different genetic behavior of the transgenes was observed among the nine GT B. napus lines. In some lines, the segregation of GT:non-GT showed a ratio of 1:1 in the BC2, BC3, and BC4 generations. However, in other GT B. napus lines the segregation ratio of GT:non-GT significantly deviated from 1:1 in the BC2 and BC3 generations, which had fewer transgenic progeny than expected, but not in the BC4 generation. Most importantly, in two GT B. napus lines the segregation of GT:non-GT did not fit into a ratio of 1:1 in the BC2, BC3 or BC4 generations due to a deficiency of transgenic progeny. For these lines, a strong reduction of transgene introgression was observed in all three B. rapa accessions. These findings imply that the genomic location of transgenes in B. napus may affect the long-term persistence of transgenes in B. rapa after hybridization has occurred.