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Nucleic Acids Research, 2003, Vol. 31, No. 18 5317-5323
© 2003 Oxford University Press

Retrovirus silencer blocking by the cHS4 insulator is CTCF independent

Shuyuan Yao1,2, Cameron S. Osborne1, Rikki R. Bharadwaj1, Peter Pasceri1, Tanya Sukonnik1, Dylan Pannell1, Félix Recillas-Targa3, Adam G. West4 and James Ellis*,1,2

1 Developmental Biology Program, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada, 2 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada, 3 Instituto de Fisiologia Celular, Universidad Nacional Autónoma de México, Departamento de Genética Molecular, México DF 04510, Mexico and 4 Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892,0540, USA

*To whom correspondence should be addressed at Room 8154, Elm Building, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Tel: +1 416 813 7295; Fax: +1 416 813 8883; Email: jellis{at}sickkids.ca
Present addresses:
Cameron S. Osborne, Laboratory of Chromatin and Gene Expression, Department of Developmental Genetics, Babraham Institute, Cambridge, UK
Adam G. West, Department of Pathology, Glasgow University, Glasgow, UK

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 LCRß-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.


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