Figure 4

Efficient and persistent YFP reporter gene expression induced by transient Cre expression in NPCs generated from Rosa26YFP transgenic mice. A-B: Long-term YFP-expression was induced after transfection of NPCs with a Cre plasmid (2 μg). Transient expression of the Cre recombinase led to successful recombination in 30% of the cells. Note that this number remains stable over time, indicating an absence of down-regulation of the reporter gene. C-F: Cre-nucleofection was non-toxic and did not change the fate of mouse NPCs. Transient expression of Cre was not toxic for the cells as shown by immunodetection of the activated form of caspase 3, an apoptotic cell marker (Casp-3A, red, C). Quantification of the number of Casp-3A positive cells 48 h after nucleofection (D) showed an increase in cell apoptosis that was not exacerbated by Cre-expression. Note that >98% of the cells are Casp-3A negative 48 hours after nucleofection. Numerous cells co-expressed the reporter gene YFP and βIII-tubulin 48 hours after Cre transfection (E). Quantification of the number of GFAP and βIII-tubulin positive cells at 48 hours post cre-transfection showed no change in the fate of differentiated NPCs (F).