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Prepublished online as a Blood First Edition Paper on September 26, 2002; DOI 10.1182/blood-2002-07-2086.
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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.
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