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Quantitative analysis of gene expression in living adult neural stem cells by gene trapping

Nature Methods volume 2, pages 363370 (2005) | Download Citation

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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.

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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

Author notes

    • Carrolee Barlow

    Present address: BrainCells Inc., 10835 Road to the Cure, San Diego, California 92121, USA.

Affiliations

  1. Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

    • John R Scheel
    • , Jasodhara Ray
    • , Fred H Gage
    •  & Carrolee Barlow

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Competing interests

C.B. is now an employee of BrainCells, Inc. No work for this manuscript was supported by funds from BrainCells, Inc.

Corresponding author

Correspondence to Carrolee Barlow.

Supplementary information

PDF files

  1. 1.

    Supplementary Table 1

    Optimizing FACS for the isolation and survival of single neural stem cells

  2. 2.

    Supplementary Table 2

    Tagged clonal populations and identity of trapped genes

  3. 3.

    Supplementary Methods

  4. 4.

    Supplementary Protocol

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DOI

https://doi.org/10.1038/nmeth755

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