Direct isolation of committed neuronal progenitor cells from transgenic mice coexpressing spectrally distinct fluorescent proteins regulated by stage-specific neural promoters.
Many tissues arise from pluripotent stem cells through cell-type specification and maturation. In the bone marrow, primitive stem cells generate all the different types of blood cells via the sequential differentiation of increasingly committed progenitor cells. Cell-surface markers that clearly distinguish stem cells, restricted progenitors, and differentiated progeny have enabled researchers to isolate these cells and to study the regulatory mechanisms of hematopoiesis. Neuronal differentiation appears to involve similar mechanisms. However, neural progenitor cells that are restricted to a neuronal fate have not been characterized in vivo, because specific cell-surface markers are not available. We have developed an alternative strategy to identify and isolate neuronal progenitor cells based on dual-color fluorescent proteins. To identify and isolate directly progenitor cells from brain tissue without the need for either transfection or intervening cell culture, we established lines of transgenic mice bearing fluorescent transgenes regulated by neural promoters. One set of transgenic lines expressed enhanced yellow fluorescent protein (EYFP) in neuronal progenitor cells and neurons under the control of the Talpha1 alpha-tubulin promoter. Another line expressed enhanced green fluorescent protein (EGFP) in immature neural cells under the control of the enhancer/promoter elements of the nestin gene. By crossing these lines we obtained mice expressing both transgenes. To isolate neuronal progenitor cells directly from the developing brain, we used flow cytometry, selecting cells that expressed EGFP and EYFP simultaneously. We expect this strategy to provide valuable material with which to study the mechanisms of neurogenesis and to develop cell-based therapies for neurological disorders.
Sawamoto
K
K
Division of Neuroanatomy, Department of Neuroscience, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Yamamoto
A
A
Kawaguchi
A
A
Yamaguchi
M
M
Mori
K
K
Goldman
S A
SA
Okano
H
H
D000818:Animals; D001921:Brain / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods*; D002490:Central Nervous System / Q000166:cytology* / Q000196:embryology; D005434:Flow Cytometry / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007381:Intermediate Filament Proteins / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D064231:Nestin; D009474:Neurons / Q000166:cytology*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism*; D019076:Transgenes; D014404:Tubulin / Q000235:genetics*
D007381:Intermediate Filament Proteins; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins