The core element of a CpG island protects avian sarcoma and leukosis virus-derived vectors from transcriptional silencing.
Unmethylated CpG islands are known to keep adjacent promoters transcriptionally active. In the CpG island adjacent to the adenosine phosphoribosyltransferase gene, the protection against transcriptional silencing can be attributed to the short CpG-rich core element containing Sp1 binding sites. We report here the insertion of this CpG island core element, IE, into the long terminal repeat of a retroviral vector derived from Rous sarcoma virus, which normally suffers from progressive transcriptional silencing in mammalian cells. IE insertion into a specific position between enhancer and promoter sequences led to efficient protection of the integrated vector from silencing and gradual CpG methylation in rodent and human cells. Individual cell clones with IE-modified reporter vectors display high levels of reporter expression for a sustained period and without substantial variegation in the cell culture. The presence of Sp1 binding sites is important for the protective effect of IE, but at least some part of the entire antisilencing capacity is maintained in IE with mutated Sp1 sites. We suggest that this strategy of antisilencing protection by the CpG island core element may prove generally useful in retroviral vectors.
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-14220 Prague 4, Czech Republic.
Filip
F
Senigl
Jirí
J
Plachý
Jirí
J
Hejnar
D000818:Animals; D001354:Avian Leukosis Virus / Q000378:metabolism*; D001665:Binding Sites; D001717:Birds; D018899:CpG Islands; D005434:Flow Cytometry; D020868:Gene Silencing; D017930:Genes, Reporter; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D052736:Rous sarcoma virus / Q000378:metabolism; D001357:Sarcoma, Avian / Q000235:genetics* / Q000821:virology*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D020079:Terminal Repeat Sequences; D014158:Transcription, Genetic
D016329:Sp1 Transcription Factor