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J. Biol. Chem., Vol. 280, Issue 2, 991-998, January 14, 2005

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Cloning and Functional Analysis of the Rhesus Macaque ABCG2 Gene

FORCED EXPRESSION CONFERS AN SP PHENOTYPE AMONG HEMATOPOIETIC STEM CELL PROGENY IN VIVO^*

Takahiro Ueda, Sebastian Brenner, Harry L. Malech, Saskia M. Langemeijer, Shira Perl, Martha Kirby¶, Oswald A. Phang, Allen E. Krouse||, Robert E. Donahue||, Elizabeth M. Kang, and John F. Tisdale**

From the Molecular and Clinical Hematology Branch, NIDDK, the Laboratory of Host Defense, NIAID, the ^¶National Human Genome Research Institute, ^||Research Court, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, and the Klinik fuer Kinder und Jugend Medizin, Universitatsklinikum Carl Gustav Carus, 01307 Dresden, Germany

Hematopoietic cells can be highly enriched for repopulating ability based upon the efflux of the fluorescent Hoechst 33342 dye by sorting for SP (side population) cells, a phenotype attributed to expression of ABCG2, a member of the ABC transporter superfamily. Intriguingly, murine studies suggest that forced ABCG2 expression prevents hematopoietic differentiation. We cloned the full-length rhesus ABCG2 and introduced it into a retroviral vector. ABCG2-transduced human peripheral blood progenitor cells (PBPCs) acquired the SP phenotype but showed significantly reduced growth compared with control. Two rhesus macaques received autologous PBPCs split for transduction with the ABCG2 or control vectors. Marking levels were similar between fractions with no discrepancy between bone marrow and peripheral blood marking. Analysis for the SP phenotype among bone marrow and mature blood populations confirmed ABCG2 expression at levels predicted by vector copy number long term, demonstrating no block to differentiation in the large animal. In vitro studies showed selective protection against mitoxantrone among ABCG2-transduced rhesus PBPCs. Our results confirm the existence of rhesus ABCG2, establish its importance in conferring the SP phenotype, suggest no detrimental effect of its overexpression upon differentiation in vivo, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications.

Received for publication, August 26, 2004 , and in revised form, October 27, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY841878.

^** To whom correspondence should be addressed: Molecular and Clinical Hematology Branch, NIDDK, National Institutes of Health, Bldg. 10, Rm. 9N116, 9000 Rockville Pike, Bethesda, MD 20892.

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