title: Expression of 2A peptide mediated tri-fluorescent protein genes were regulated by epigenetics in transgenic sheep.
abstract: A number of gene therapy applications and basic research would benefit from vectors expressing multiple genes. In this study, we constructed 2A peptide based tricistronic lentiviral vector and generated transgenic lambs by injecting lentivirus carrying the tricistronic vector into perivitelline space of zygotes. Of 7 lambs born, 2 lambs (#6 and #7) carried the transgene. However, no fluorescent proteins were identified in transgenic sheep. To investigate why the transgene was silenced in transgenic sheep, we analyzed the methylation status of transgene. The methylation level of CMV promoter was 76.25% in #6, and 64.7% in #7. In the coding region of three fluorescent protein genes, methylation levels were extremely high, with the average level of 98.3% in #6 and 98.4% in #7 respectively. Furthermore, the ratio of GFP(+) cells were increased significantly when the fibroblasts derived from the transgenic sheep were treated with 5-azaC and/ or TSA. Our results showed that 2A peptide based tricistronic construct was subjected to hypermethylation in transgenic sheep. Moreover, the silencing could be relieved by treating with methytransferase inhibitor and/or deacetylase inhibitor.
authors.affiliation: College of Animal Science and Technology, Shihezi University, Shihezi, China.
authors.forename: Yongzhi
authors.initials: Y
authors.lastname: Tian
authors.forename: Wenrong
authors.initials: W
authors.lastname: Li
authors.forename: Liqin
authors.initials: L
authors.lastname: Wang
authors.forename: Chenxi
authors.initials: C
authors.lastname: Liu
authors.forename: Jiapeng
authors.initials: J
authors.lastname: Lin
authors.forename: Xuemei
authors.initials: X
authors.lastname: Zhang
authors.forename: Ning
authors.initials: N
authors.lastname: Zhang
authors.forename: Sangang
authors.initials: S
authors.lastname: He
authors.forename: Juncheng
authors.initials: J
authors.lastname: Huang
authors.forename: Bin
authors.initials: B
authors.lastname: Jia
authors.forename: Mingjun
authors.initials: M
authors.lastname: Liu
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019175:DNA Methylation; D017931:DNA Primers; D044127:Epigenesis, Genetic; D005434:Flow Cytometry; D020868:Gene Silencing; D008164:Luminescent Proteins / Q000235:genetics*; D010455:Peptides / Q000235:genetics*; D012756:Sheep; D019076:Transgenes
chemical_list: D017931:DNA Primers; D008164:Luminescent Proteins; D010455:Peptides