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Simple and Efficient Isolation of Hematopoietic Stem Cells from H2K-zFP Transgenic Mice

^a Department of Life Science, Swiss Federal Institute of Technology, Lausanne, Switzerland;
^b The Burnham Institute, La Jolla, California, USA;
^c Department of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California, USA

Key Words. Hematopoietic stem cells • Fluorescent protein • Prospective isolation

Correspondence: Alexey V. Terskikh, Assistant Professor, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, USA. Telephone: 858-646-3100 (ext. 3624); Fax: 858-713-6274; e-mail: Terskikh{at}Burnham.org

We have generated a transgenic mouse line that allows for simple and highly efficient enrichment for mouse hematopoietic stem cells (HSCs). The transgene expresses a green fluorescent protein variant (zFP) under the control of H2K^b promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs express exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2K-zFP mice. Up to 90% of zFP^bright cells are also c-kit^high, Sca-1^high, Lin^neg, Flk-2^neg, which is a bona fide phenotype for long-term HSCs. Double-sorted zFP^bright HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2K-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs without using fluorescence-conjugated monoclonal antibodies. This approach will greatly facilitate gene transfer, including short interfering RNA for gene knockdown, into HSCs and, consequently, into all other hematopoietic lineages.

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