eGFP reporter genes silence LCRbeta-globin transgene expression via CpG dinucleotides.
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.
Dalle
Bruno
B
Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Rubin
Joel E
JE
Alkan
Ozan
O
Sukonnik
Tanya
T
Pasceri
Peter
P
Yao
Shuyuan
S
Pawliuk
Rob
R
Leboulch
Philippe
P
Ellis
James
J
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
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005914:Globins