A new red fluorescent protein that allows efficient marking of murine hematopoietic stem cells.
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.
Sanuki
Seiko
S
Advanced Biomedical Applications, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Hamanaka
Sanae
S
Kaneko
Shin
S
Otsu
Makoto
M
Karasawa
Satoshi
S
Miyawaki
Atsushi
A
Nakauchi
Hiromitsu
H
Nagasawa
Toshiro
T
Onodera
Masafumi
M
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
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein