The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites.
Retroviruses are highly susceptible to transcriptional silencing and position effects imparted by chromosomal sequences at their integration site. These phenomena hamper the use of recombinant retroviruses as stable gene delivery vectors. As insulators are able to block promoter-enhancer interactions and reduce position effects in some transgenic animals, we examined the effect of an insulator on the expression and structure of randomly integrated recombinant retroviruses. We used the cHS4 element, an insulator from the chicken beta-like globin gene cluster, which has been shown to reduce position effects in transgenic Drosophila. A large panel of mouse erythroleukemia cells that bear a single copy of integrated recombinant retroviruses was generated without using drug selection. We show that the cHS4 increases the probability that integrated proviruses will express and dramatically decreases the level of de novo methylation of the 5' long terminal repeat. These findings support a primary role of methylation in the silencing of retroviruses and suggest that cHS4 could be useful in gene therapy applications to overcome silencing of retroviral vectors.
Rivella
S
S
Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Callegari
J A
JA
May
C
C
Tan
C W
CW
Sadelain
M
M
D020413:3' Untranslated Regions / Q000235:genetics; D016475:3T3 Cells; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D002460:Cell Line; D019175:DNA Methylation; D003850:Deoxyribonuclease I / Q000378:metabolism; D005434:Flow Cytometry; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid; D020079:Terminal Repeat Sequences; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D016662:Virus Integration; D014779:Virus Replication
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D014764:Viral Proteins; D005914:Globins; D003850:Deoxyribonuclease I