title: Reactivation of transgene expression by alleviating CpG methylation of the Rous sarcoma virus promoter in transgenic quail cells.
abstract: In this study, we investigated the relative expression of the Rous sarcoma virus (RSV) promoter-driven 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reduced the CpG methylation status in the RSV promoters of the QEFs (86.42 to 48.41%); even after 5-azadC was withdrawn, CpG methylation remained decreased in expanded culture (16.28%).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.
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, 599 Gwanak-ro, Gwanak-gu Seoul 151-921, Korea. wkd316@snu.ac.kr
authors.forename: Hyun-Jun
authors.initials: HJ
authors.lastname: Jang
authors.forename: Jin Won
authors.initials: JW
authors.lastname: Choi
authors.forename: Young Min
authors.initials: YM
authors.lastname: Kim
authors.forename: Sang Su
authors.initials: SS
authors.lastname: Shin
authors.forename: Kichoon
authors.initials: K
authors.lastname: Lee
authors.forename: Jae Yong
authors.initials: JY
authors.lastname: Han
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000166:cytology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000821:virology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D011533:Proviruses / Q000187:drug effects / Q000235:genetics; D011784:Quail / Q000235:genetics*; D052736:Rous sarcoma virus / Q000187:drug effects / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001374:Azacitidine