Effect of electroporation conditions on loach sperm for successful gene transfer and early development

We used leach Misgurnus anguillicaudatus sperm to study the effect of the electroporation conditions for fish sperm on the capacity of transferring a foreign gene into the eggs and the development of their derivative embryos. Results showed that sperm electroporated at low field strengths resulted in a higher hatching rate and a lower abnormality rate, compared to the high field strength group. However, the success rate of gene transfer of the low field strength group was lower than that of the high field strength group. Sperm electroporated at a concentration of 175 mu g/ml plasmid DNA had a higher gene transfer rate than did those at 100 mu g/ml or lower. The hatching rate of embryos derived from ova fertilized with sperm treated at 250 mu g/ml DNA decreased abruptly, even at a low field strength (3.5 kV). Increasing the number of pulses per cycle (up to 2(11)) and the number of cycles (up to 12) enhanced the success rate of gene transfer. Dot and Southern blot analyses and PCR detection proved the existence of transferred DNA in embryos following sperm-electroporation. An optimal condition of electroporation for leach sperm to obtain normal development in transgenic leach is suggested.