Figure 6.

PBase-removable insertion DNA (pRIND) can be used as a selection donor for COIN and excised in an inducible and scarless manner. (A) Schematic representation of pRIND-PuroΔTK donor construct and primers used to assay for insertion. (B) PuroΔTK-SDS constructs were targeted to the Rosa26 locus by the inclusion of 500bp homology arms and used to stimulate integration of a GFP expression cassette at the Lef1 locus. The percent of GFP+ cells was measured by flow cytometry after 18 days. (C) Three clones treated with or without 4-OHT were treated with FIAU and percent surviving clones is shown. (D) PCR using the External, Int/Ext, and Internal primers denoted in A to measure insertion and excision of the pRIND cassette for a representative clone. (E) Sequencing of the Cas9-sgRNA cut site generated using the External Primer amplicons from D using nested sequencing primers. The scarless removal of pRIND can be traced by sequencing before and after excision by tamoxifen (OHT) treatment. The schematic at right demonstrates the anticipated amplicons available for sequencing analysis.