Pseudorabies Virus-Based Gene Delivery to Rat Embryonic Spinal Cord Grafts

Abstract

The construction and application of recombinant pseudorabies viruses (PrVs) for the delivery of beta-galactosidase and/or green fluorescent protein (GFP) genes to rat embryonic spinal cord cells are reported here. These viruses were specifically designed to infect embryonic spinal cord neurons, which can be grafted into a lesioned spinal cord in order to restore the lost functions of the host cord. The recombinant viruses were constructed in two steps. The small subunit of the ribonucleotide reductase (RR) gene was first abolished by a frameshift mutation and an expression cassette containing the lacZ gene alone or together with the GFP gene was then inserted in place of the early protein 0 (EP0) gene of PrV. The reporter gene cassettes were positioned downstream from the PrV latency-associated promoter. Using an ex vivo system, we infected embryonic spinal cord explants with these viruses and found that neither vRREP0lac nor vRREP0lacgfp exerted any cytotoxic effect at all. It was also revealed that these viruses infect embryonic cells with high efficiency, and that infected neurons grafted into the spinal cord express the inserted reporter genes for periods of up to 12 weeks. This system offers a new approach for foreign gene transfer to neurons grafted into the CNS.