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Journal of Virology, July 1999, p. 5490-5496, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Amelioration of Retroviral Vector Silencing in Locus Control Region beta -Globin-Transgenic Mice and Transduced F9 Embryonic Cells

Cameron S. Osborne,1 Peter Pasceri,1 Rakesh Singal,2,dagger Tanya Sukonnik,1 Gordon D. Ginder,2,3 and James Ellis1,4,*

Programs in Developmental Biology and Blood and Cancer Research, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X81; Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada4; Department of Medicine, Division of Medical Oncology, University of Minnesota, Minneapolis, Minnesota 55455-03622; and Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298-00373

Received 17 December 1998/Accepted 9 April 1999

Retroviral vectors are transcriptionally silenced in hematopoietic stem cells, and this phenomenon must be overcome for effective gene therapy of blood diseases. The murine stem cell virus (MSCV) vector completely silences beta -globin reporter genes regulated by locus control region (LCR) elements 5'HS2 to 5'HS4 in seven of eight transgenic mice. Here, we show that no single known MSCV silencer element is sufficient for complete LCR beta -globin transgene silencing. However, partial silencing of high-copy transgenes is conveyed by the MSCV direct repeat and promoter elements. The CpG methylation pattern of silenced and expressed MSCV promoter transgenes is virtually identical, demonstrating that silencing does not absolutely correlate with methylation status. Combined mutations in all four MSCV silencer elements leads to expression of beta -globin in 6 of 10 transgenic mice. The same mutations incorporated into the HSC1 retrovirus vector direct neo gene expression in 71% of transduced F9 embryonic carcinoma cells. These studies demonstrate that combined mutation of four retroviral silencer elements relieves complete silencing in most transgenic mice and transduced F9 cells and suggests that novel silencer elements remain. Enhanced expression of the HSC1 vector in primitive stem cells is well suited for blood gene therapy applications.

* Corresponding author. Mailing address: Program in Developmental Biology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8. Phone: (416) 813-7295. Fax: (416) 813-8883. E-mail: jellis{at}sickkids.on.ca.

dagger Present address: Section of Hematology and Medical Oncology, LSU Medical Center, Shreveport, LA 71130-3932.

Journal of Virology, July 1999, p. 5490-5496, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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