title: Simple and efficient isolation of hematopoietic stem cells from H2K-zFP transgenic mice.
abstract: 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 H2Kb 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.
authors.affiliation: Department of Life Science, Swiss Federal Institute of Technology, Lausanne, Switzerland.
authors.forename: Didier
authors.initials: D
authors.lastname: Surdez
authors.forename: Beatrice
authors.initials: B
authors.lastname: Kunz
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Wagers
authors.forename: Irving L
authors.initials: IL
authors.lastname: Weissman
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Terskikh
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000737:chemistry; D002460:Cell Line; D002469:Cell Separation; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006183:H-2 Antigens / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic / Q000502:physiology; D019076:Transgenes
chemical_list: D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D049452:Green Fluorescent Proteins