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"Rainbow" Reporters for Multispectral Marking and Lineage Analysis of Hematopoietic Stem Cells

^a Flow Cytometry Facility and
^b Department of Hematopoiesis, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland, USA;
^c Flow Cytometry Core Laboratory, Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA;
^d Department of Anatomy and Cell Biology, The George Washington University Medical Center, Washington, DC, USA

Key Words. Fluorescent protein reporter genes • Multiparameter flow cytometry • Oncoretroviral and lentiviral vectors • Gene transfer • Hematopoietic stem cells

Robert G. Hawley, Ph.D., Department of Hematopoiesis, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, Maryland 20855, USA. Telephone: 301-738-0424; Fax: 301-738-0444; e-mail: hawleyr{at}usa.redcross.org

Hematologic diseases potentially benefiting from gene-based therapies involving hematopoietic stem cells (HSCs) include hereditary hemoglobinopathies, immunodeficiency syndromes, and congenital bleeding disorders such as hemophilia A, as well as acquired diseases like AIDS. Successful treatment of these blood diseases with gene-modified HSCs requires high efficiency gene delivery to the target cell population and persistence of transgene expression following differentiation. We review flow cytometric procedures that permit simultaneous, noninvasive measurements of transgene expression and phenotypic discrimination of hematopoietic cell subsets. Central to this approach has been the recent development of a spectrum of blue, cyan, and yellowish-green fluorescent reporters based on the jellyfish Aequorea victoria green fluorescent protein and the discovery of a red fluorescent protein in Discosoma coral. This methodology should facilitate the optimization of oncoretroviral and lentiviral vectorology and HSC transduction protocols for the ultimate purpose of HSC-directed gene therapy.

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