title: eGFP reporter genes silence LCRbeta-globin transgene expression via CpG dinucleotides.
abstract: beta-Globin transgenes regulated by the locus control region (LCR) are dominantly silenced by linked bacterial reporter genes in transgenic mice. Enhanced green fluorescent protein (eGFP) from jellyfish is an alternative reporter used in retrovirus vectors to transfer LCRbeta-globin genes into bone marrow. We show here that the eGFP coding sequence silences LCRbeta-globin in transgenic mice, but the PGK promoter did not provoke such silencing. As eGFP contains 60 CpG dinucleotides, which are targets of DNA methylation, we synthesized a novel CpG-free variant called dmGFP. Its utility was demonstrated in MSCV retrovirus vectors transcriptionally controlled by the viral 5'LTR or internal PGK or EF1alpha promoter. Specific fluorescence was detected from eGFP, and at lower levels from dmGFP, in transduced mouse CFU-S and embryonic stem cells. While eGFP was rarely silenced in CFU-S, dmGFP was not silenced in these progenitors. Moreover, the dmGFP coding sequence did not silence LCRbeta-globin in transgenic mice, showing that the eGFP silencing mechanism acts primarily via CpG dinucleotides. However, LCRbeta-globin expression remained suboptimal, indicating that other silencing pathways recognize dmGFP in the absence of CpG dinucleotides. We conclude that dmGFP ameliorates silencing, but optimal LCRbeta-globin expression is obtained in the absence of nonmammalian reporters.
authors.affiliation: Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Bruno
authors.initials: B
authors.lastname: Dalle
authors.forename: Joel E
authors.initials: JE
authors.lastname: Rubin
authors.forename: Ozan
authors.initials: O
authors.lastname: Alkan
authors.forename: Tanya
authors.initials: T
authors.lastname: Sukonnik
authors.forename: Peter
authors.initials: P
authors.lastname: Pasceri
authors.forename: Shuyuan
authors.initials: S
authors.lastname: Yao
authors.forename: Rob
authors.initials: R
authors.lastname: Pawliuk
authors.forename: Philippe
authors.initials: P
authors.lastname: Leboulch
authors.forename: James
authors.initials: J
authors.lastname: Ellis
mesh_terms: D000818:Animals; D001483:Base Sequence; D018899:CpG Islands / Q000235:genetics / Q000502:physiology*; D018900:Dinucleotide Repeats / Q000235:genetics / Q000502:physiology; D020868:Gene Silencing; D017930:Genes, Reporter / Q000235:genetics; D005914:Globins / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D019901:Locus Control Region / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation; D019076:Transgenes
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005914:Globins