Figure 1.

Generation of PITX3-IRES2-tTA knock-in mice. A, The schematic diagram depicts the generation of PITX3-IRES2-tTA knock-in mice in which the IRES2-tTA expression cassette was inserted into the SacII site of PITX3 exon 4 immediately after the stop codon (asterisk) of the PITX3 gene. B, Southern blot of genomic DNA isolated from mouse embryonic stem (ES) cell clones shows a correct gene targeting of the PITX3 gene locus in ES cell clones 1E2 and 1F1. As predicted, the insertion of the IRES2-tTA-LNL gene-targeting cassette into the PITX3 gene locus generated a new 3.7 kb AflII fragment in clones 1E2 and 1F1 (arrowhead). C, A PCR assay shows the correct modification of the PITX3 allele through amplification of genomic DNA prepared from mouse tail biopsy. D, The quantification of PITX3 mRNA expression in the midbrain tissues dissected from 6-month-old nTg and PITX3-IRES2-tTA (tTA) heterozygous knock-in mice. The level of PITX3 mRNA expression was measured by qRT-PCR. Six or more mice were used for each genotype. E, The schematic diagram depicts the generation of PITX3-IRES2-tTA/tetO-H2Bj-GFP (H2GFP) double-transgenic mice. Sample images show the distribution of the nuclear localized H2Bj/GFP fusion protein in the midbrain coronal sections of 1-month-old H2GFP double-transgenic mice. The mDA neurons in both the SNC and VTA were revealed by immunostaining with an antiserum against TH. Scale bars: top, 100 μm; bottom, 50 μm. F, H2GFP (green) and TH (red) costaining in different brain regions. Scale bar, 100 μm.