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 inve rtebrate physiological solution was as good as that of the control gro up. The fertilization rate reached as high as 94.7-99.6% (93.0-99.7% f or 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 cy cles. Moreover, the fertilization rate of sperm electroporated in the presence of foreign DNA (opAFP-2000CAT) ranging from 0.1 to 3.2 mu g a nd at voltages ranging from 2 to 10 kV, at 2(7) or 2(11) pulses for si x or 12 cycles showed no differences from the control sperm. After DNa se digestion, the genome of the electroporated sperm was analysed by p olymerase chain reaction, and it was shown that a 138-bp product was a mplified corresponding to the transgene's amplification product. South ern blotting also showed that a positive band located at the same posi tion as that of opAFP-2000CAT was found in the electroporated sperm af ter DNase treatment. Analysis by PCR of the genome isolated from a tro chophore-stage abalone larva, derived from sperm electroporated with 3 .2 mu g opAFP-2000CAT; showed the existence of foreign DNA in 13 out o f 20 examined samples (65%). The integration of the transferred DNA in to the genome of transgenic abalone was also shown by Southern blot an alysis. Furthermore, CAT activity was positive for the experimental la rvae, but the level of CAT expression was lower than that of larvae de rived from sperm electroporated with pCAT-Control vector, driven by SV 40 promoter and enhancer sequences. These results demonstrate the pote ntial for the use of sperm as mass gene transfer strategy in marine mo llusks such as abalone.