title: Enhanced green fluorescent protein targeted to the Sca-1 (Ly-6A) locus in transgenic mice results in efficient marking of hematopoietic stem cells in vivo.
abstract: OBJECTIVE
  Hematopoietic stem cells are important clinically, both as targets of disease and as reagents for cellular therapy. Studies in hematopoietic stem cell biology have been hampered by difficulties in purifying and manipulating these cells. To facilitate these studies, we sought to develop a system for targeting genes of interest to the hematopoietic stem cell compartment in transgenic mice.
  
  
  METHODS
  We used Sca-1, a glycosyl phosphatidylinositol-anchored protein expressed on the surface of all hematopoietic stem cells in commonly used inbred mouse strains. We created a mutant Sca-1 allele in which the enhanced green fluorescent protein (EGFP) cDNA is integrated into the Sca-1 locus by homologous recombination in embryonic stem cells.
  
  
  RESULTS
  EGFP protein is detectable in all hematopoietic tissues of mice heterozygous for the mutant Sca-1 allele. Growth and development of these mice are normal. No adverse effects of long-term, high-level EGFP expression were noted. Sca-1 positive cells coexpress EGFP in all tissues and lineages examined, as predicted by the targeting strategy. Sca-1 and EGFP expression are coordinately up-regulated in splenocytes from mutant mice. The Lin(-)EGFP(+) bone marrow population contains all progenitor activity in Sca-1(+)(/EGFP) mice. The Lin(-)EGFP(+) bone marrow cells are equivalent to Lin(-)Sca-1(+) cells in long-term repopulation and serial transplantation assays.
  
  
  CONCLUSIONS
  The hematopoietic stem cell compartment appears to be targeted in Sca-1(+)(/EGFP) mutant mice. This system should be useful for studying the normal biology of hematopoietic stem cells and for targeting other genes to this cellular compartment.
authors.affiliation: Department of Internal Medicine, Division of Oncology, Stem Cell Biology Section, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
authors.forename: Piia
authors.initials: P
authors.lastname: Hanson
authors.forename: Vikram
authors.initials: V
authors.lastname: Mathews
authors.forename: Sarah H
authors.initials: SH
authors.lastname: Marrus
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Graubert
mesh_terms: D000818:Animals; D000950:Antigens, Ly / Q000235:genetics*; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D003584:Cytological Techniques / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics* / Q000254:growth & development; D013154:Spleen / Q000166:cytology / Q000378:metabolism
chemical_list: D000950:Antigens, Ly; D008164:Luminescent Proteins; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D049452:Green Fluorescent Proteins