12915_2020_815_MOESM1_ESM.pdf (135.83 kB)

Additional file 1 of Real-time tracking of stem cell viability, proliferation, and differentiation with autonomous bioluminescence imaging

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posted on 2020-07-04, 03:50 authored by Michael Conway, Tingting Xu, Andrew Kirkpatrick, Steven Ripp, Gary Sayler, Dan Close
Additional file 1: Fig. S1. Luciferin:luciferase component integration and transcriptional expression ratios in autobioluminescent cells following extended time in culture. PDF File detailing the results of qPCR and qRT-PCR experiments to determine luciferin:luciferase integration and expression post-transfection. (a) Genomic DNA from iPSC-lux lines 11 passages after stable transfection with a 30:1 molar ratio of Stem-luxCDEF:Stem-luxAB or cardiomyocytes stably transfected with the tetracycline-repressible lux operon (TET-lux; 1:1 molar ratio), were probed by qPCR to determine the actual gene expression ratios post-transfection. Because the luciferin and luciferase pathway genes were expressed using 2A elements to concatenate each component into a single open reading frame, the second gene of each operon was used for qPCR analysis. As described in [12], this approach provides an average expression level for each operon while accounting for possible reduced expression of the genes distal to the promoter. (b) qRT-PCR analysis reveals that, despite their 27:1 genomic integration ratio, the luciferin pathway is only transcribed at 15:1 relative to the luciferase pathway. Data is available at [15].


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