Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons

摘要

We have generated embryonic stem cells and transgenic mice with green fluorescentudprotein (GFP) inserted into the Pitx3 locus via homologous recombination. In the centraludnervous system, Pitx3 directed GFP was visualised in dopaminergic (DA) neurons in theudsubstantia nigra and ventral tegmental area. Live primary DA neurons can be isolated byudfluorescence-activated cell sorting from these transgenic mouse embryos. In culture,udPitx3-GFP is co-expressed in a proportion of ES-derived DA neurons. Furthermore, stemudcell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors andudwere sensitive to DA specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. Weudanticipate that the Pitx3-GFP ES cells could be used as a powerful model system forudfunctional identification of molecules governing mDA neuron differentiation and for preclinicaludresearch including pharmaceutical drug screen and transplantation. The Pitx3udknock-in mice, on the other hand, could be used for purifying primary neurons forudmolecular studies associated with the midbrain-specific DA phenotype at a level notudpreviously feasible. These mice would also provide a useful tool to study DA fateuddetermination from embryo- or adult-derived neural stem cells.