GENE DELIVERY INTO PRIMARY CEREBRAL CORTICAL NEURONS BY LENTIVIRAL VECTOR

Objectives: The goals of this study were to assess the transduction efficiency and the kinetic process of EGFP-expressing lentiviral vectors in highly purified rat cortical neuron cultures.

Methods: Rat embryonic cerebral cortical neurons were cultured for eight days prior to infection with lentiviral vectors at a multiplicity of infection (m.o.i.) of 10, 20, or 40. The virus-containing medium was removed 2h later. After 2, 3, and 4 days (DIV), The percentage of EGFP-expressing cells was calculated by FACS analysis. Three days after infection, cultures were fixed in 4% paraformaldehyde for immunofluorescence.

Results: At a given virus concentration, the percentage of EGFP-expressing neurons increased at 3 DIV compared with that at 2 DIV. However, there were no significant differences between the percentage of EGFP-expressing neurons at 3 and 4 DIV. In addition, the percentage of EGFP-expressing neurons increased with an m.o.i. of 20, 40 compared with m.o.i. of 10. But there was no significant difference between the percentage of EGFPexpression neurons at m.o.i.=20 and 40. The efficacy of transduction is as a function of the time in culture and of the virus dose. The highest percentage of EGFP-expression achieved was 46.77% at 4 DIV with an m.o.i. of 20.

Conclusion: he results show that lentiviral vectors are not only good prospects for in vivo gene delivery, but are also good candidates for in vitro studies of the function of gene products in cerebral cortical neurons.