Life Technologies

Detection of transgene expression With the use of the CytoTune®-iPS Sendai Reprogramming Kit, the viral transgenes that are delivered to the somatic cells at the time of reprogramming are eventually cleared from the reprogrammed cells, allowing for virus-free iPSCs. To confirm their absence, RNA was isolated from all six donor samples at three different time points: the initial fibroblast lines, the cells at day 7 post–CytoTune® transduction, and the isolated iPSC clones at various passages. The TaqMan® iPSC Sendai Detection Kit was used to detect the presence and level of expression of exogenous transcription factors Dilution of individual transcription factors for Ctrl-1 Relative expression 10,000,000 Fibroblast Day 7 Post-transduction Passage 13 iPSC Passage 15 iPSC 1,000,000 100,000 10,000 10 (Oct3/4, Sox2, Klf4, and c-Myc) delivered to cells, as well as the presence of Sendai virus RNA. The assays in the TaqMan® iPSC Sendai Detection Kit do not detect the corresponding endogenous factors. As expected, cells harvested at day 7 post-transduction displayed high levels of expression of the transgenes, while the fibroblasts and iPSC clones were free of these genes (Figure 8). We observed that the timing of virus clearance varied from line to line and even clone to clone from the same line, presumably due to the difference in the proliferation rate of these cells. Clearance was observed as early as passage 10 (p10) in one clone and as late as passage 15 (p15) in another (Figure 9). After testing the expression levels of the 4 exogenous factors, we observed that c-Myc was the last factor to be cleared in most lines. 1 0.1 p10 OCT SeV SOX SeV KLF SeV Myc SeV Figure 8. Clearance of all four Sendai reprogramming factors detected by qPCR. iPSCs were cultured in KSR-supplemented iPSC medium on MEFs for 4 days. Cells were harvested using TrypLE ™ enzyme and stored in RNAlater ® solution before RNA isolation using the RNAqueous®-4PCR Kit and cDNA synthesis using SuperScript® VILO® Master Mix. Expression levels were measured using the TaqMan® iPSC Sendai Detection Kit and QuantStudio™ 12K Flex Real-Time PCR System, and the threshold cycle values were evaluated to determine the relative expression of exogenous transcription factor genes (OCT3/4, SOX2, KLF4, and c-MYC) and Sendai virus (not shown) in Ctrl-1 fibroblasts, Ctrl-1 cells from day 7 post-transduction, Ctrl-1 iPSC clone 10 at passage 13, and Ctrl-1 iPSC clone 10 at passage 15. As expected, at day 7 post-transduction, significant amounts of exogenous transcription factors were expressed. Expression levels decreased dramatically once the specific iPSC clone was established, and then became reduced to the fibroblast level (free of Sendai virus) at passage 15. 21 Life Technologies | Parkinson's cell model p11 p12 p13 p14 p15 PD-1 Clone S1 Clear PD-1 Clone 10 X – – – – – Ctrl-1 – – – X – Clear PD-2 – Clear PD-3 X Clear MSA – Clear Ctrl-2 – – Clear Figure 9. Clearance of exogenous reprogramming factors and Sendai virus. The TaqMan® iPSC Sendai Detection Kit was used to determine the presence of Sendai virus and the exogenous genes OCT3/4, SOX2, KLF4, and c-MYC for all six iPSC lines generated in this study. Clear: Sendai virus and the exogenous genes not detected. X: Sendai virus detected, not cleared. –: not tested.

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