Abstract
The potential of neural stem cells (NSCs) for the treatment of neurodegenerative diseases makes the identification and characterization of genes involved in neural stem cell responses therapeutically important. Although technologies exist for measuring gene expression in cells, they often provide only a representative expression profile specific to a stimulus and time. We developed a complementary technology based on a retroviral-vector gene-trap approach that uses β-lactamase–induced disruption of fluorescence resonance energy transfer in the fluorophore CCF-2/AM. A library of 'tagged' adult rat NSCs was generated by transduction with gene-trap virus produced from a single-integrant packaging cell line that allowed us to quantitatively analyze dynamic gene expression changes in real time in living NSCs. Using this library we identified previously unknown genes regulated by oxidative stress, indomethacin and factors that induce differentiation, and show that one of the trapped genes, Sox6, is sufficient to induce astrocytic differentiation when overexpressed.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Accession codes
References
Ray, J., Peterson, D.A., Schinstine, M. & Gage, F.H. Proliferation differentiation, and long-term culture of primary hippocampal neurons. Proc. Natl. Acad. Sci. USA 90, 3602–3606 (1993).
Gage, F.H. Mammalian neural stem cells. Science 287, 1433–1438 (2000).
Markakis, E.A. & Gage, F.H. Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J. Comp. Neurol. 406, 449–460 (1999).
Kokaia, Z. & Lindvall, O. Neurogenesis after ischaemic brain insults. Curr. Opin. Neurobiol. 13, 127–132 (2003).
Jin, K. et al. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc. Natl. Acad. Sci. USA 98, 4710–4715 (2001).
Parent, J.M., Vexler, Z.S., Gong, C., Derugin, N. & Ferriero, D.M. Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann. Neurol. 52, 802–813 (2002).
Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z. & Lindvall, O. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat. Med. 8, 963–970 (2002).
Takahashi, K. & Tanaka-Kunishima, M. Monitoring early neuronal differentiation by ion channels in ascidian embryos. J. Neurobiol. 37, 3–22 (1998).
Suhonen, J.O., Peterson, D.A., Ray, J. & Gage, F.H. Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature 383, 624–627 (1996).
Modo, M. et al. Transplantation of neural stem cells in a rat model of stroke: assessment of short-term graft survival and acute host immunological response. Brain Res. 958, 70–82 (2002).
Modo, M. et al. Tracking transplanted stem cell migration using bifunctional, contrast agent-enhanced, magnetic resonance imaging. Neuroimage 17, 803–811 (2002).
Geschwind, D.H. et al. A genetic analysis of neural progenitor differentiation. Neuron 29, 325–339 (2001).
Karsten, S.L. et al. Global analysis of gene expression in neural progenitors reveals specific cell-cycle, signaling, and metabolic networks. Dev. Biol. 261, 165–182 (2003).
Zlokarnik, G. et al. Quantitation of transcription and clonal selection of single living cells with β-lactamase as reporter. Science 279, 84–88 (1998).
Zlokarnik, G. Fusions to β-lactamase as a reporter for gene expression in live mammalian cells. Methods Enzymol. 326, 221–244 (2000).
Somia, N.V., Zoppe, M. & Verma, I.M. Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery. Proc. Natl. Acad. Sci. USA 92, 7570–7574 (1995).
Whitney, M. et al. A genome-wide functional assay of signal transduction in living mammalian cells. Nat. Biotechnol. 16, 1329–1333 (1998).
Gage, F.H., Ray, J. & Fisher, L.J. Isolation, characterization, and use of stem cells from the CNS. Annu. Rev. Neurosci. 18, 159–192 (1995).
Frohman, M.A., Dush, M.K. & Martin, G.R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002 (1988).
Zambrowicz, B.P. et al. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature 392, 608–611 (1998).
Hansen, J. et al. A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome. Proc. Natl. Acad. Sci. USA 100, 9918–9922 (2003).
Barlow, C. et al. Loss of the ataxia-telangiectasia gene product causes oxidative damage in target organs. Proc. Natl. Acad. Sci. USA 96, 9915–9919 (1999).
Pong, K. Oxidative stress in neurodegenerative diseases: therapeutic implications for superoxide dismutase mimetics. Expert Opin. Biol. Ther. 3, 127–139 (2003).
Day, B.J., Patel, M., Calavetta, L., Chang, L.Y. & Stamler, J.S. A mechanism of paraquat toxicity involving nitric oxide synthase. Proc. Natl. Acad. Sci. USA 96, 12760–12765 (1999).
Monje, M.L., Toda, H. & Palmer, T.D. Inflammatory blockade restores adult hippocampal neurogenesis. Science 302, 1760–1765 (2003).
Monje, M.L. & Palmer, T. Radiation injury and neurogenesis. Curr. Opin. Neurol. 16, 129–134 (2003).
Narahara, M. et al. Immunocytochemical localization of beta-citryl-L-glutamate in primary neuronal cells and in the differentiation of P19 mouse embryonal carcinoma cells into neuronal cells. Biol. Pharm. Bull. 23, 1287–1292 (2000).
Hamada-Kanazawa, M. et al. Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid. FEBS Lett. 560, 192–198 (2004).
Townley, D.J., Avery, B.J., Rosen, B. & Skarnes, W.C. Rapid sequence analysis of gene trap integrations to generate a resource of insertional mutations in mice. Genome Res. 7, 293–298 (1997).
Zhang, B., Schmoyer, D., Kirov, S. & Snoddy, J. GOTree Machine (GOTM): a web-based platform for interpreting sets of interesting genes using Gene Ontology hierarchies. BMC Bioinformatics 5, 16 (2004).
Keppler, D., Leier, I., Jedlitschky, G. & Konig, J. ATP-dependent transport of glutathione S-conjugates by the multidrug resistance protein MRP1 and its apical isoform MRP2. Chem. Biol. Interact. 111–112, 153–161 (1998).
Maack, C. et al. Oxygen free radical release in human failing myocardium is associated with increased activity of rac1-GTPase and represents a target for statin treatment. Circulation 108, 1567–1574 (2003).
Miller, A.C. & Samid, D. Tumor resistance to oxidative stress: association with ras oncogene expression and reversal by lovastatin, an inhibitor of p21ras isoprenylation. Int. J. Cancer 60, 249–254 (1995).
Geyer, J., Doring, B., Failing, K. & Petzinger, E. Molecular cloning and functional characterization of the bovine (Bos taurus) organic anion transporting polypeptide Oatp1a2 (Slco1a2). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 137, 317–329 (2004).
Smits, P. et al. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev. Cell 1, 277–290 (2001).
Smits, P. & Lefebvre, V. Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell survival and development of the nucleus pulposus of intervertebral discs. Development 130, 1135–1148 (2003).
Murakami, A., Ishida, S., Thurlow, J., Revest, J.M. & Dickson, C. SOX6 binds CtBP2 to repress transcription from the Fgf-3 promoter. Nucleic Acids Res. 29, 3347–3355 (2001).
Nakashima, K. & Taga, T. Mechanisms underlying cytokine-mediated cell-fate regulation in the nervous system. Mol. Neurobiol. 25, 233–244 (2002).
Palmer, T.D., Markakis, E.A., Willhoite, A.R., Safar, F. & Gage, F.H. Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J. Neurosci. 19, 8487–8497 (1999).
Hsieh, J. et al. IGF-I instructs multipotent adult neural progenitor cells to become oligodendrocytes. J. Cell Biol. 164, 111–122 (2004).
Somia, N.V., Kafri, T. & Verma, I.M. Piecing together more efficient gene expression. Nat. Biotechnol. 17, 224–225 (1999).
Barlow, C. et al. Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell 86, 159–171 (1996).
Sandberg, R. et al. Regional and strain-specific gene expression mapping in the adult mouse brain. Proc. Natl. Acad. Sci. USA 97, 11038–11043 (2000).
Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).
Acknowledgements
We thank R. Tsien for his helpful discussions and support; B. Miller for her guidance in NSC culture; and L. Moore, A. Willhoite, the members of the flow cytometry core facility and members of the Barlow lab for their advice. We also thank M.L. Gage and G.P. Scheel for their editorial comments; J. Simon for figure preparation; Aurora Biosciences (Panvera) for providing the CCF-2/AM and the p237 vector; R. Barnard and J. Rainey of the Young lab (The Salk Institute) for assistance with the plate reader; and T. Palmer for his suggestions during the preparation of this manuscript. This work was supported by NS039601-04, the Lookout Fund, the Frederick B. Rentschler Developmental Chair (C.B.), NIH fellowship F31 NS10860-01 (J.R.S.) and the Vi and John Alder Chair (F.H.G.).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
C.B. is now an employee of BrainCells, Inc. No work for this manuscript was supported by funds from BrainCells, Inc.
Supplementary information
Supplementary Table 1
Optimizing FACS for the isolation and survival of single neural stem cells (PDF 43 kb)
Supplementary Table 2
Tagged clonal populations and identity of trapped genes (PDF 95 kb)
Rights and permissions
About this article
Cite this article
Scheel, J., Ray, J., Gage, F. et al. Quantitative analysis of gene expression in living adult neural stem cells by gene trapping. Nat Methods 2, 363–370 (2005). https://doi.org/10.1038/nmeth755
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth755