Reactivation of transgene expression by alleviating CpG methylation of the Rous sarcoma virus promoter in transgenic quail cells

In this study, we investigated the relative expression of the Rous sarcoma virus (RSV) promoterdriven expression of enhanced green fluorescent protein (EGFP) in fibroblasts of transgenic quails. We analyzed the direct influence of CpG methylation of the RSV promoter on the transcriptional activity of delivered transgenes. Embryonic fibroblasts collected from homozygous transgenic quail (TQ2) were treated with 50 μM of DNA methyltransferase inhibitor followed by 5-aza-2'-deoxycytidine (5-azadC) for 48 h, and changes in expression were then analyzed by flow cytometry. The results show a significant increase of EGFP expression in TQ2 embryonic fibroblasts (QEFs) (2.64% to 79.84%). Subsequent methylation-specific amplification revealed that 5-azadC significantly reduced the CpG methylation status in the RSV promoters of the QEFs (86.42 to 48.41%); even after 5-azadC was withdrawn, CpG methylation remained decreased in expanded culture (16.28%). Further analysis showed that potential transcription factor binding sites existed in the CpG methylation site of the RSV promoter. These results may provide the basis for understanding the epigenetic mechanism responsible for transgenic animal production and genetic preservation.