Electroporation of sperm to introduce foreign DNA into the genome of loach (Misgurnus anguillicaudatus)

Abstract

An antifreeze protein gene promoter from ocean pout (Macrozoarces americanus) linked to a chinook salmon (Oncorhynchus tshawytscha) growth hormone cDNA (opAFPGHc) was introduced into loach (Misgurnus anguillicaudatus) sperm via electroporation under the following conditions: amplitude, 8 kV; burst time, 1.6 s; number of pulses; 28; pulse time, 120 μs; cycle, 6. Compared with the untreated sperm, the fertilization rate driven by the electroporated sperm was decreased slightly, with no significant effect on hatchability. The success rate of gene transfer was 50%. Genomic Southern blotting demonstrated that DNA from transgenic loach contained positive band(s) with higher (37.5%), the same, or lower molecular size as the transferred opAFPGHc fragment. There was a mosaic distribution of exogenous DNA in a wide variety of tissues analyzed. The average weight gain between the 14th and 161th day of the experimental group was 2.5-fold higher than those of the control and mock-treated groups. The body length distribution of the experimental group was rather widely scattered instead of being normally distributed.