O-10: Sperm Mediated Gene Transfer Using Adjuvant Preserving Fertility for Production of Transgenic Chicken Expressing

Background: Low uptake of exogenous DNA by sperm and reduced number of fertilized oocyte by transfected sperm are the major obstacles for progression of sperm mediated gene transfer. Therefore, the modification of sperm mediated gene transfer procedure needs to be required. The purpose of this study was to evaluate the efficiency of FuGene 6 compare to lipofection in transfection medium for internalizing of the foreign gene in the rooster sperm cell. Materials and Methods: Semen were washed in Beltsville extender for removal of the seminal plasma by repetitive washing. Then sperm (1 ×109 sperm/ml) was incubated with exogenous DNA (Enhanced Green Fluorescent Protein, EGFP) in Beltsville extender containing: 1. no additive, 2. lipofectamine 1000 (1:1), and 3. FuGene 6 (1:1). Transfection was measured using PCR reaction in the sperm cell and new born chickens after artificial insemination. Motility, viability, membrane integrity, acrosome integrity and their fertilizing ability using artificial insemination were considered as index of sperm quality post transfection. Results: Although there was not significant different in rate of transfection in sperm cell between Lipofectamin (11%) and FuGene 6 (10%), they were significantly higher compare to Control group (2%). The higher significant percentage of motility, viability and fertility rate were observed in FuGene 6 (29.1 ± 1.5, 33.5 ± 2.4, 26.8 ± 1.8, respectively) compare to control group (10.5 ± 1.5, 12.8 ± 2.4, 14.9 ± 1.8) and Lipofectamine (13.5 ± 1.5, 16.1 ± 2.4, 17.3 ± 1.8). Moreover, the higher significant percentage of transfection in chickens were observed in FugGen 6 (33.5%) rather than control (14%) and lipofectamine (16%) groups. For membrane integrity and acrosome integrity, no significant different was observed for all groups. Conclusion: Our data revealed that adding FuGene 6 would resulte in higher rate of sperm quality after transfection which led to higher rate of fertility and production of transgenic chicken.