Figure 6. Schemes to generate transgene-free iPSCs.
Scheme A (black arrows): After transfection of transposon and transposase DNA into MEFs to generate primary iPSCs, individual iPSC colonies are picked to identify colonies with 2 copy transposon integrations, by Southern blot analysis. These clones are then expanded and the transposase is re-expressed to remove the transposons. HSVtk-FIAU negative selection is used to identify integration-free iPSCs. Scheme B (red arrows): Using an optimized protocol, analysis of individual clones can be bypassed. Primary iPSCs can be pooled and directly subjected to transposon removal and HSVtk-FIAU selection.