Study on factors of the transfection efficiency and the optimum conditions for the sperm-mediated gene transfer by electroporation in goats

To establish the basis for production of transgenic goats using the sperm transfecting exogenous DNA by electroporation.The sperm viability was examined by counting the died sperms on the blood corpuscle counter under microscope and the positive sperms which were digested or not after electroporation were detected by situ-hybridization to investigate the factors affecting DNA transfection of goat sperms by electroporation and in order to obtain the optimized electroporation procedure and condition.The results showed that(1) Electroporation could be used to transfect exogenous DNA into the goat spermatozoon,because the existence of exogenous DNA in the cytoplasm or nucleus of transfected sperm after digestion with DNAase I was detected.(2) Electric field intensity,electroporation time and frequency of electroporation affected the sperm viability.There was no significant influence on the binding of exogenous DNA to sperms by electric field intensity and electroporation time.But the frequency of electroporation significantly affected internalization of exogenous DNA into sperms.The optimized conditions of electroporation for goat sperms were 400V,200μs,and 1 treament.Under these conditions,the viability,the transfection rates of sperms digested or undigested with DNAaseⅠwere 0.45,27.6%,22.5%,respectively.(3) The transfection rate after digestion with DNAaseⅠ could be enhanced 14% by washing the sperms before electroporating.Furthermore,by combining the methods of washing sperms with incubation with foreign DNA before electroporating,the transfection rate increased by 5.8%.(4) The transfection rate was not different between goat donors and the transfection rates of No.1,2,3 goat were 22.5%,23.2%,25.3%,respectively.In addition,the distribution of internalized DNA in the transfected sperms was irregular.The results indicated that the optimized conditions for exogenous DNA transfer by electroporation were: washing the sperms thoroughly and then co-incubating them with exogenous DNA before electroporating under the condition of 400V,200μs,and 1 treament.