Sperm as a carrier to introduce an exogenous DNA fragment into the oocyte of Japanese abalone (Haliotis divorsicolor suportexta)

We investigated gene transfer in abalone via electroporated sperm. The mobility of sperm electroporated either in seawater or in marine invertebrate physiological solution was as good as that of the control group. The fertilization rate reached as high as 94.7-99.6% (93.0-99.7% for the control group) when 200 eggs were fertilized by 10(6) or 10(7) sperm treated with electroporation al 10 kV and 2(7) pulses for six cycles. Moreover, the fertilization rate of sperm electroporated in the presence of foreign DNA (opAFP-2000CAT) ranging from 0.1 to 3.2 mu g and at voltages ranging from 2 to 10 kV, at 2(7) or 2(11) pulses for six or 12 cycles showed no differences from the control sperm. After DNase digestion, the genome of the electroporated sperm was analysed by polymerase chain reaction, and it was shown that a 138-bp product was amplified corresponding to the transgene's amplification product. Southern blotting also showed that a positive band located at the same position as that of opAFP-2000CAT was found in the electroporated sperm after DNase treatment. Analysis by PCR of the genome isolated from a trochophore-stage abalone larva, derived from sperm electroporated with 3.2 mu g opAFP-2000CAT; showed the existence of foreign DNA in 13 out of 20 examined samples (65%). The integration of the transferred DNA into the genome of transgenic abalone was also shown by Southern blot analysis. Furthermore, CAT activity was positive for the experimental larvae, but the level of CAT expression was lower than that of larvae derived from sperm electroporated with pCAT-Control vector, driven by SV40 promoter and enhancer sequences. These results demonstrate the potential for the use of sperm as mass gene transfer strategy in marine mollusks such as abalone.