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Figure 2 | Stem Cell Research & Therapy

Figure 2

From: Excision of viral reprogramming cassettes by Cre protein transduction enables rapid, robust and efficient derivation of transgene-free human induced pluripotent stem cells

Figure 2

Assessment of TAT-Cre-mediated transgene deletion efficiency. (A) Genomic polymerase chain reaction (PCR) for the confirmation of transgene deletion. Three individual clones were treated with 0.5, 1 or 2 μM TAT-Cre for 5 hours and PCR was performed using primers against WPRE and β-actin. F, AR1034ZIMA loxP-modified iPSC line clone 1 (fl-ARiPSC); NC, negative (water) control; WPRE, viral element; BA, β-actin. (B) Genomic PCR for the confirmation of transgene deletion in a polyclonal population. fl-ARiPSCs were treated with 0.5, 1 or 2 μM TAT-Cre for 5 hours. Cells were expanded polyclonally and PCR was performed using primers against WPRE and β-actin. (C) Validation of genomic PCR analysis. Genomic DNA from loxP-modified and transgene deleted cells were mixed to create defined dilutions as given. (D) Quantification of transgene-deleted clones using different concentrations and time duration of TAT-Cre. (E) ARiPS-Cre reporter cell line was treated with 2 μM Cre protein to validate recombination efficiency. Cre-mediated recombination induced the expression of green fluorescence protein (EGFP), by deleting the loxP-flanking RFP gene. Scale bar: 120 μm.

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Stem Cell Research & Therapy

ISSN: 1757-6512