Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo (original) (raw)

Nature Protocols volume 1, pages 3049–3055 (2006)Cite this article

Abstract

Single-cell genetic manipulation in an intact brain environment is an informative approach to study molecular mechanism of adult neurogenesis. Here, we describe a protocol for retrovirus-mediated single-cell gene knockout in adult new neurons in vivo. A gene of interest is disrupted in adult floxed mice by a vector based on the Moloney murine leukemia retrovirus, expressing Cre recombinase. High-titer retrovirus is prepared by transfecting plasmids into the HEK293T cells and by concentrating the supernatant containing virus. The retrovirus is stereotaxically injected into the dentate gyrus. Cre recombinase is transduced and expressed in a small fraction of adult new neurons in an intact environment, and the gene knockout is highly efficient within the transduced neurons. Virus preparation takes 7 days, but virus injections take less than 1 h per mouse. By changing the survival time of the mice after the injection, one can analyze the effects on new neurons at different ages.

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Author notes

  1. Ayumu Tashiro
    Present address: Centre for the Biology of Memory, Norwegian University of Science and Technology, Medical-Technical Research Centre, NO-7489, Trondheim, Norway

Authors and Affiliations

  1. Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, 92037, California, USA
    Ayumu Tashiro, Chunmei Zhao & Fred H Gage

Authors

  1. Ayumu Tashiro
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  2. Chunmei Zhao
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  3. Fred H Gage
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Correspondence toFred H Gage.

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Tashiro, A., Zhao, C. & Gage, F. Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo.Nat Protoc 1, 3049–3055 (2006). https://doi.org/10.1038/nprot.2006.473

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