title: A new red fluorescent protein that allows efficient marking of murine hematopoietic stem cells.
abstract: BACKGROUND
  Genetic marking of hematopoietic stem cells (HSCs) with multiple fluorescent proteins (FPs) would allow analysis of their features, including interaction with adjacent cells. However, there are few red FPs that are comparable to green FPs in terms of low toxicity and high fluorescent intensity. This study has evaluated the usefulness of Kusabira Orange (KO) originated from the coral stone Fungia concinna as a red FP for marking of HSCs
  
  
  METHODS
  A vector used was the MSCV-type retroviral vector, D Delta Nsap that has the PCC4 cell-passaged myeloproliferative sarcoma virus derived long terminal repeat devoid of a binding site for YY1 and the primer-binding site derived from the dl587rev, respectively. The vector was cloned with the codon-optimized KO cDNA for higher expression in mammalian cells (huKO) and converted to the corresponding retroviruses pseudotyped with the vesicular stomatitis virus G envelope protein, then transduced into c-KIT(+)Sca-1(+)Lineage(-) cells obtained from C57BL/6 (Ly5.1) mice followed by transplantation into lethally irradiated Ly5.2 mice.
  
  
  RESULTS
  Approximately 70% of donor-derived cells highly expressed huKO at 16 weeks post-transplantation. Furthermore, the high expression of huKO was also detected in serially transplanted mice, suggesting that expression of huKO per se had little deleterious effect on murine hematopoiesis. In double marking experiments, huKO-expressing hematopoietic cells were easily distinguished from those expressing EGFP by flow cytometry and fluorescent microscope analysis.
  
  
  CONCLUSIONS
  Overall, the results obtained from the present study suggest that huKO can be used as a valuable and versatile red fluorescent marker for HSCs.
authors.affiliation: Advanced Biomedical Applications, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
authors.forename: Seiko
authors.initials: S
authors.lastname: Sanuki
authors.forename: Sanae
authors.initials: S
authors.lastname: Hamanaka
authors.forename: Shin
authors.initials: S
authors.lastname: Kaneko
authors.forename: Makoto
authors.initials: M
authors.lastname: Otsu
authors.forename: Satoshi
authors.initials: S
authors.lastname: Karasawa
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Nakauchi
authors.forename: Toshiro
authors.initials: T
authors.lastname: Nagasawa
authors.forename: Masafumi
authors.initials: M
authors.lastname: Onodera
mesh_terms: D000818:Animals; D019070:Cell Lineage; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D018076:DNA, Complementary; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein