P-151: Optimal Condition of IntroducingGreen Fluorescent Protein (GFP) Geneinto The Rainbow Trout (Oncorhynchusmykiss) Sperm by Electroporation Method

Background: Many techniques have been developed to increase DNA molecules into sperms called sperm mediated gene transfer (SMGT) and one of them is electroporation. Sperm mediated gene transfer is an ideal method for mass gene transfer in organisms with external fertilization. Gene transfer is applied to rainbow trout sperm and the effects of electroporation on sperms and rate of gene uptake by sperms were examined. Materials and Methods: In this study, sperms were collected from ten male rainbow trout at the age of 3 years. Through the method of hand stripping, average of 1.5-5 ml sperms were collected from each; then sperms were counted and the amount always have been diluted to 2×106/μl. The condition of electroporation such as field strength pulse duration, medium in use, concentration of gene, and effect of incubation after electroporation were optimized. Current study has many groups with variety of conditions in each stage. Therefore, before each stage, the group and condition have been explained. Sperms were electroporated in the existence of linear pIRES2-EGFP plasmid at 50-100 ng/μl, with field strength between 100 to 1500 v/cm. Results: Present studies indicated a direct relationship between DNA concentration and rate of gene uptake and also pulse length and intensity of gene uptake. By the way, if electroporated sperm/ gene was incubated after electroporation, the rate of gene uptake will be increased. On the other hand, if the seminal plasma is used as diluent, the viability and gene uptake will be significantly increased. Conclusion: Compared to the non-electroporated group, these result showed that electroporation enhanced the capacity of spermatozoa to uptake plasmid DNA so that ultra violet microscope showed 93.5% of electroporated sperm (in the best and optimal condition) absorbed the plasmid DNA with different intensity.