AIM: To prepare the transgenic donor cells for developing new breed of transgenic pig against Japanese encephalitis virus.MATERIALS AND METHIODS: The isolation and passage culture of the fibroblasts from the skin tissue in the ear and belly of a newborn pig were performed by using the direct adherent culture method.The 5th generation fibroblasts were harvested and transfected with siRNA-encoding plasmid against Japanese encephalitis virus and then fluorescence in situ hybridization (FISH), RT-PCR and immunofluorescence assay (IA) were performed to identify whether the target sequence was transferred successfully into pig fibroblasts.RESULTS: The positive signals for the target sequence were detected in the nucleus of the porcine fibroblast by using FISH, which demonstrated that the target cDNA has integrated into porcine genome and retained its function in replication.Flow cytometric analysis showed that its integration rate was 4%.The positive bands for the target sequence were observed by using RT-PCR, which confirmed that the target sequence retain its function in transcription.The positive signals for the target sequence were detected within the cytoplasm of the porcine fibroblast by using IA, which indicated that the target sequence retain its function in translation.CONCLUSION: We have successfully obtained the transgenic donor cells for developing new breed of transgenic pig against Japanese encephalitis virus.