Efficient marking of neural stem cell-derived neurons with a modified murine embryonic stem cell virus, MESV2

Treatments for nervous system disorders that involve transplanting genetically modified neural stem cells may ultimately be feasible. As a step towards this therapeutic approach, a novel murine embryonic stem cell gammaretroviral vector was developed with features designed to optimize transgene expression in neural stem cells and to increase vector safety. All potential start sites of translation in the 5' leader were removed. These sites may compete with an inserted transgene for translation initiation, and also produce potentially immunogenic peptides. Further, all of the gag gene sequences were replaced with a well-defined constitutive transport element from avian leukemia virus to promote nuclear export of viral RNA, and to eliminate any homology between the vector and a murine leukemia virus-derived gag-pol packaging plasmid. Two versions of the virus were made in which EGFP expression was driven either by the Rous sarcoma virus U3 enhancer or by a combination of sequences from the Syn1 and Pgk-1 promoters. Both of these viruses efficiently transduced neural stem cells isolated from embryonic rat hippocampus, and robust EGFP expression was observed in neurons derived from these cells following differentiation in vitro.