title: Retrovirus silencer blocking by the cHS4 insulator is CTCF independent.
abstract: Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRbeta-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.
authors.affiliation: Developmental Biology Program, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
authors.forename: Shuyuan
authors.initials: S
authors.lastname: Yao
authors.forename: Cameron S
authors.initials: CS
authors.lastname: Osborne
authors.forename: Rikki R
authors.initials: RR
authors.lastname: Bharadwaj
authors.forename: Peter
authors.initials: P
authors.lastname: Pasceri
authors.forename: Tanya
authors.initials: T
authors.lastname: Sukonnik
authors.forename: Dylan
authors.initials: D
authors.lastname: Pannell
authors.forename: Félix
authors.initials: F
authors.lastname: Recillas-Targa
authors.forename: Adam G
authors.initials: AG
authors.lastname: West
authors.forename: James
authors.initials: J
authors.lastname: Ellis
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D000076246:CCCTC-Binding Factor; D002645:Chickens; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005914:Globins / Q000235:genetics; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D012097:Repressor Proteins / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics; D012190:Retroviridae / Q000235:genetics
chemical_list: D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D018626:Retroelements; D005914:Globins; D004247:DNA