Fig. 3. SNCA triplication THtdTomato reporter generation and quality control to facilitate purification and live-cell imaging of dopaminergic neurons.

a Schematic demonstrating iPSC source, generation, modification, and differentiation with tdTomato reporter permitting identification and isolation of dopaminergic neurons. Previously described iPSC line derived from a patient with Parkinson’s Disease caused by triplication of the SNCA locus resulting in four copies of the gene encoding α-synuclein. iPSC line was then modified with tyrosine hydroxylase:tdTomato reporter¹⁴. Adapted from Hallaci et al. 2022²⁵. b–f Quality control and validation of SNCA triplication THtdTomato reporter line including 5’ and 3’ PCR products to confirm proper insertion. b Agarose gel stained with ethidium bromide to demonstrate examples of seven clones that contain the expected PCR products (626 bp product confirmed proper insertion of the 5’ end of the reporter construct and an 878 bp product confirming proper insertion at the 3’ end). PCR reactions run separately but combined into the same wells of the agarose gel for each clone to visualize clones passing and failing PCR quality control. A subset of clones have a single larger band and these are excluded from further testing. Band length was reproduced in an additional PCR from clones showing proper size to evaluate by Sanger sequencing. c Sanger sequencing of PCR products in (b) confirming correct insertion of tdTomato cassette. d G-banded karyotype (performed by WiCell) confirms normal karyotype in modified clone. e Example of live imaging of endogenous THtdTomato fluorescence at 10x. Neurons with this live imaging morphology and appearance are consistently obtained from multiple differentiations (greater than ten) from this cell line. f Immunofluorescence co-localization of Rabbit (Rbt) anti-RFP and Sheep (Sh) anti-tyrosine hydroxylase visualized with Alexa Fluor 546 donkey anti-rabbit and Alexa Fluor 488 donkey anti-sheep, respectively. Colocalization of anti-RFP and anti-tyrosine hydroxylase staining reproduces in greater than three differentiations in the cell line used for these experiments. Source data are provided as a Source Data file.