Efficient gene transfer in mouse neural precursors with a bicistronic retroviral vector

Gene transfer into neural precursors is a powerful approach to study the fu nction of specific gene products during nervous system development. Here we describe a retrovirus-based methodology to transduce foreign genes into mo use neural precursors. We used a high-titer bicistronic retroviral vector t hat encodes a marker gene, placental alkaline phosphatase (plap), and a sel ection gene, neomycin phosphotransferase II (neoR), under the translational control of two retroviral internal ribosome entry segments. Transduction e fficiency even without selection was up to 95% for multipotential neurosphe res derived from embryonic striata and grown with basic fibroblast growth f actor 2. Expression of plap and neoR was sustained with time in culture and upon differentiation into neurons, astrocytes, and oligodendrocytes, as sh own by double immunofluorescence labeling with cell type-specific markers, Western blotting, and neomycin resistance. However, levels of plap were dec reased in differentiated oligodendrocytes. Transduction with the same vecto r of neonatal oligodendrocyte precursors grown in oligospheres consistently resulted in a lower proportion of plap-immunoreactive cells and enhanced c ell death in the absence of neomycin. However, plap expression was maintain ed in some differentiated oligodendrocytes expressing galactocerebroside or myelin basic protein. In that neurospheres can be easily expanded in vitro and factors enabling their differentiation into the three main central ner vous system cell types are being elucidated, this methodology could be used in the future to produce large number of transduced, differentiated neural cells. J. Neurosci. Res. 65:208-219, 2001. (C) 2001 Wiley-Liss, Inc.