"Rainbow" reporters for multispectral marking and lineage analysis of hematopoietic stem cells
T S Hawley1, W G Telford, R G Hawley
1Flow Cytometry Facility, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855, USA. Hawleyr@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.
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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