The use of an adeno-associated viral vector for efficient bicistronic expression of two genes in the CNS

This chapter describes the cloning of a bicistronic AAV vector that uses the foot and mouth disease virus 2A sequence to efficiently express two genes from a single promoter. Bicistronic expression of a therapeutic gene and a reporter gene is desirable so that the axons from transduced neurons can be tracked and, after CNS injury, the amount of axonal sprouting or regeneration quantified. We go on to describe how to perform a pyramidotomy model of CNS injury and the injection of AAV vectors into the sensorimotor cortex to provide efficient transduction and bicistronic gene expression in cortical neurons such that transduced axons are detectable in the dorsal columns of the spinal cord.

• 1
  The 2A peptide sequence is only 24 amino acids long and can mediate efficient bicistronic expression of two gene products from a single promoter by undergoing ribosomal skipping (referred to as "self-cleavage") during protein translation.
• 2
  Theoretically, the two gene products are expressed in a 1:1 ratio and in practice each gene product is expressed at a high level.
• 3
  The 2A and 2A-like bicistronic systems have been shown to be highly effective in the CNS where high expression levels of both genes have been demonstrated