Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons.
We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3-GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3-GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells.
Zhao
Suling
S
Institute for Stem Cell Research, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh, United Kingdom, EH9 3JQ, UK.
Maxwell
Sarah
S
Jimenez-Beristain
Antonio
A
Vives
Joaquim
J
Kuehner
Eva
E
Zhao
Jiexin
J
O'Brien
Carmel
C
de Felipe
Carmen
C
Semina
Elena
E
Li
Meng
M
D000818:Animals; D002478:Cells, Cultured; D004298:Dopamine / Q000096:biosynthesis* / Q000235:genetics; D004622:Embryo, Mammalian; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D008636:Mesencephalon / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011247:Pregnancy; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
D018398:Homeodomain Proteins; D008164:Luminescent Proteins; D014157:Transcription Factors; C433629:homeobox protein PITX3; D049452:Green Fluorescent Proteins; D004298:Dopamine