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Prepublished online as a Blood First Edition Paper on September 26, 2002; DOI 10.1182/blood-2002-07-2086. This Article Full Text Full Text (PDF) All Versions of this Article: 2002-07-2086v1 101/4/1603    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Bharadwaj, R. R. Articles by Ellis, J. Search for Related Content PubMed PubMed Citation Articles by Bharadwaj, R. R. Articles by Ellis, J. Related Collections Red Cells Gene Expression Gene Therapy Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 February 2003, Vol. 101, No. 4, pp. 1603-1610 RED CELLS LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of -globin intron-2 Rikki R. Bharadwaj, Cecelia D. Trainor, Peter Pasceri, and James Ellis From the Developmental Biology Program, Hospital for Sick Children, Toronto, ON, Canada; the Department of Molecular and Medical Genetics, University of Toronto, Toronto, ON, Canada; and the Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD. Human -globin transgenes regulated by the locus control region (LCR) express at all integration sites in transgenic mice. For such LCR activity at ectopic sites, the 5'HS3 element requires the presence of the AT-rich region (ATR) in -globin intron-2. Here, we examine the dependence of 5'HS3 LCR activity on transcription factor binding sites in the ATR. In vitro DNaseI footprint analysis and electrophoretic mobility shift assays of the ATR identified an inverted double Gata-1 site composed of 2 noncanonical sequences (GATT and GATG) and an Oct-1 consensus site. Mutant Oct-1, Gata-1, or double mutant sites were created in the ATR of the BGT50 construct composed of a 5'HS3 /-globin hybrid transgene. Transgenes with double mutant sites expressed at all sites of integration, but mean expression levels in transgenic mice were reduced from 64% per copy (BGT50) to 37% (P < .05). Mutation of the inverted double Gata-1 site had no effect at 61% per copy expression levels. In contrast, mutation of the Oct-1 site alone reduced per-copy expression levels to 31% (P < .05). We conclude that the ability of 5'HS3 to activate expression from all transgene integration sites is dependent on sequences in the ATR that are not bound at high affinity by transcription factors. In addition, the Oct-1 site in the ATR is required for high-level 5'HS3 /-globin transgene expression and should be retained in LCR-globin expression cassettes designed for gene therapy. © 2003 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: A. Buzina, M. I. Aladjem, J. L. Kolman, G. M. Wahl, and J. Ellis Initiation of DNA replication at the human {beta}-globin 3' enhancer Nucleic Acids Res., August 5, 2005; 33(14): 4412 - 4424. [Abstract] [Full Text] [PDF] V. E. H. Wang, T. Schmidt, J. Chen, P. A. Sharp, and D. Tantin Embryonic Lethality, Decreased Erythropoiesis, and Defective Octamer-Dependent Promoter Activation in Oct-1-Deficient Mice Mol. Cell. Biol., February 1, 2004; 24(3): 1022 - 1032. [Abstract] [Full Text] [PDF]

	Copyright © 2003 by American Society of Hematology         Online ISSN: 1528-0020