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Blood, 1 July 2001, Vol. 98, No. 1, pp. 65-73
GENE THERAPY
Lack of neighborhood effects from a transcriptionally active
phosphoglycerate kinase-neo cassette located between the
murine  -major and -minor globin genes
Richard M. Kaufman,
Zhi
Hong Lu,
Rajesh Behl,
Jo M. Holt,
Gary K. Ackers, and
Timothy J. Ley
From the Departments of Pathology/Laboratory Medicine,
the Division of Oncology, Section of Stem Cell Biology, and the
Department of Biochemistry, Washington University School of Medicine,
St Louis, MO.
For the treatment of  -globin gene defects, a homologous
recombination-mediated gene correction approach would provide
advantages over random integration-based gene therapy strategies.
However, "neighborhood effects" from retained selectable marker
genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the 6 position of
the mouse -major globin gene (6I) as a step toward
the development of a murine model system that could serve as a platform
for therapeutic gene correction studies. The marked -major gene can
be tracked at the level of DNA, RNA, and protein, allowing
investigation of the impact of a retained phosphoglycerate kinase
(PGK)-neo cassette located between the mutant -major and -minor
globin genes on expression of these 2 neighboring genes. Although the
PGK-neo cassette was expressed at high levels in adult erythroid cells,
the abundance of the 6I mRNA was indistinguishable from
that of the wild-type counterpart in bone marrow cells. Similarly, the
output from the -minor globin gene was also normal. Therefore, in
this specific location, the retained, transcriptionally active PGK-neo
cassette does not disrupt the regulated expression of the adult
-globin genes.
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