Abstract:Abstract Human β-defensin 3 (hBD3) gene mammary special expressing vector named pEBB containing EGFP report gene was transfected into bovine fetal fibroblasts by Lipofectmine TM2000 and G418 selection (600 μg/mL) was applied to select positive cells. After identified with PCR, these positive cell clones were donated to produce transgenetic cloned embryo with somatic cell nuclear transplantation followed by monitoring the existence of hBD3 gene in embryo genome using PCR. After tranplanting these trangenetic cloned embryos into 64 receptors' uterus, there were 21 cows pregnant 3 months later. This research provides feasible techniques for getting trans human β-defensin 3(hBD3) transgenetic bovine embryos, producing transgenetic cows and proventing cow mastitis.