TY - JOUR
T1 - "color Timer" mice
T2 - Visualization of neuronal differentiation with fluorescent proteins
AU - Kanki, Hiroaki
AU - Shimabukuro, Marilia Kimie
AU - Miyawaki, Atsushi
AU - Okano, Hideyuki
N1 - Funding Information:
We are grateful to Dr. Masahiro Yamaguchi (U. of Tokyo; Japan) for the nestin promoter-enhancer construct; to Molecular Biological Laboratories (MBL; Nagoya, Japan) for the KOr cDNA and the numerical data for the KOr spectra; to the GENSAT Project for the DCX-EGFP BAC Tg mice; and to the “Tamago Club” of the Research Resource Center of RIKEN BSI for microinjection of the nestin/KO transgene DNA fragment. We are indebted to Yoichi “Yotan” Imaizumi for his advice in regard to the immunohistochemical analyses, to Sadafumi Suzuki and Dr. Yumi Matsuzaki for their help with cell sorting, to Chikako Hara for her help with preparing Additional file 1, and to Satoshi Suyama, François Renault-Mihara, and Chikako Hara for their advice in regard to time-lapse imaging. We also thank other members of the Okano and Miyawaki laboratories, in particular the members of the Adult Neurogenesis Group ("Minashigos”) of the former for discussions, advice, encouragement, and/or entertainment throughout this study. This work has been supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT; to HO, to HK) and from RIKEN BSI (to AM).
PY - 2010
Y1 - 2010
N2 - The molecular mechanisms governing the differentiation of neural stem cells (NSCs) into neuronal progenitor cells and finally into neurons are gradually being revealed. The lack of convenient means for real-time determination of the stages of differentiation of individual neural cells, however, has been hindering progress in elucidating the mechanisms. In order to be able to easily identify the stages of differentiation of neural cells, we have been attempting to establish a mouse system that would allow progression of neuronal differentiation to be visualized based on transitions between fluorescence colors by using a combination of mouse genetics and the ever-expanding repertoire of fluorescent proteins. In this study we report the initial version of such a mouse system, which we call "Color Timer." We first generated transgenic (Tg; nestin/KOr Tg) mice in which production of the fluorescent protein Kusabira-Orange (KOr) is controlled by the gene regulatory elements within the 2nd intronic enhancer of the nestin gene, which is a good marker for NSCs, so that NSCs would emit orange fluorescence upon excitation. We then confirmed by immunohistochemical and immunocytochemical analyses that the KOr fluorescence closely reflected the presence of the Nestin protein. We also confirmed by a neurosphere formation assay that the intensity of the KOr fluorescence correlated with "stemness" of the cell and it was possible to readily identify NSCs in the two neurogenic regions, namely the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle, in the brain of adult nestin/KOr Tg mice by the orange fluorescence they emitted. We then crossed nestin/KOr mice with doublecortin-enhanced Green Fluorescent Protein Tg mice, whose immature neurons emit green fluorescence upon excitation, and it was possible to visualize the progress of NSC-to-neuron differentiation by the transition between fluorescence colors from orange to green. This two-color initial version of the "Color Timer" mouse system will provide a powerful new tool for neurogenesis research.
AB - The molecular mechanisms governing the differentiation of neural stem cells (NSCs) into neuronal progenitor cells and finally into neurons are gradually being revealed. The lack of convenient means for real-time determination of the stages of differentiation of individual neural cells, however, has been hindering progress in elucidating the mechanisms. In order to be able to easily identify the stages of differentiation of neural cells, we have been attempting to establish a mouse system that would allow progression of neuronal differentiation to be visualized based on transitions between fluorescence colors by using a combination of mouse genetics and the ever-expanding repertoire of fluorescent proteins. In this study we report the initial version of such a mouse system, which we call "Color Timer." We first generated transgenic (Tg; nestin/KOr Tg) mice in which production of the fluorescent protein Kusabira-Orange (KOr) is controlled by the gene regulatory elements within the 2nd intronic enhancer of the nestin gene, which is a good marker for NSCs, so that NSCs would emit orange fluorescence upon excitation. We then confirmed by immunohistochemical and immunocytochemical analyses that the KOr fluorescence closely reflected the presence of the Nestin protein. We also confirmed by a neurosphere formation assay that the intensity of the KOr fluorescence correlated with "stemness" of the cell and it was possible to readily identify NSCs in the two neurogenic regions, namely the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle, in the brain of adult nestin/KOr Tg mice by the orange fluorescence they emitted. We then crossed nestin/KOr mice with doublecortin-enhanced Green Fluorescent Protein Tg mice, whose immature neurons emit green fluorescence upon excitation, and it was possible to visualize the progress of NSC-to-neuron differentiation by the transition between fluorescence colors from orange to green. This two-color initial version of the "Color Timer" mouse system will provide a powerful new tool for neurogenesis research.
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UR - http://www.scopus.com/inward/citedby.url?scp=77249156870&partnerID=8YFLogxK
U2 - 10.1186/1756-6606-3-5
DO - 10.1186/1756-6606-3-5
M3 - Article
C2 - 20205849
AN - SCOPUS:77249156870
SN - 1756-6606
VL - 3
JO - Molecular brain
JF - Molecular brain
IS - 1
M1 - 5
ER -