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A dual promoter lentiviral vector for the in vivo evaluation of gene therapeutic approaches to axon regeneration after spinal cord injury

Gene Therapy volume 17, pages 577–591 (2010)Cite this article

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Abstract

The identification of axon growth-promoting genes, and overexpression of these genes in central nervous system (CNS) neurons projecting to the spinal cord, has emerged as one potential approach to enhancing CNS regeneration. Assessment of the regenerative potential of candidate genes usually requires axonal tracing of spinal projections, ideally limited to neurons that express the candidate gene. Alternatively, coexpression of a reporter gene such as enhanced green fluorescent protein (GFP) from an internal ribosomal entry site can be used to identify neurons expressing the candidate gene, but this strategy does not label corticospinal axons in the spinal cord. We therefore developed a dual promoter lentiviral vector in which a potentially therapeutic transgene is expressed from the cytomegalovirus-enhanced chicken β-actin promoter and the fluorescent protein copGFP is expressed from the elongation factor-1α promoter. The vector was constructed to be compatible with the Gateway recombination system for efficient introduction of transgenes through entry shuttle vectors. We show both simultaneous expression of a candidate and reporter gene in corticospinal and red nucleus neurons, and efficient labeling of their axons after lesions in the cervical spinal cord. This expression system is therefore an accurate and efficient means of screening candidate genes in vivo for enhancement of axonal growth.

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Acknowledgements

We thank Dr M Tessier-Lavigne for kindly providing the plasmid encoding for the myristoylated cytoplasmic domain of DCCΔP1.

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  1. K Löw

    Present address: 3Current address: Neurodegenerative Studies Laboratory, Brain Mind Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.,

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  1. Department of Neurosciences, University of California-San Diego, La Jolla, CA, USA

    K Löw, A Blesch, J Herrmann & M H Tuszynski

  2. Veterans Affairs Medical Center, San Diego, CA, USA

    M H Tuszynski

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Löw, K., Blesch, A., Herrmann, J. et al. A dual promoter lentiviral vector for the in vivo evaluation of gene therapeutic approaches to axon regeneration after spinal cord injury. Gene Ther 17, 577–591 (2010). https://doi.org/10.1038/gt.2010.14

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