Transposon copy number in the RMCE-in HEK, HeLa, and murine ES cell clones.

<p><b>(A)</b> Southern blot of selected genomic DNA from <i>KpnI</i> digested HEK, (<b>B)</b> HeLa and <i>PstI</i> digested (<b>C)</b> mES clones. A 700bp dCTP³² labeled RFP probe (red rectangle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161471#pone.0161471.g001" target="_blank">Fig 1A</a>) identified transposition of the gene cassette at bands > 2900bp as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161471#pone.0161471.g001" target="_blank">Fig 1A</a>. Clone numbers are depicted above each lane.</p

Genomic mapping of chosen clones.

<p>Long-distance inverse PCR was used to map the position of the RMCE docking site integration in HEK colony 1.5 and 5.3, HeLa colony 2.7 and 3.6 <b>(A)</b>, and mES colony II-F1, II-H2, and I-F10 <b>(B).</b> Plasmid backbone (grey) and 5’ LIR sequences (purple) obtained by Sanger sequencing are shown above the area of insertion.</p

Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System - Table 1

<p>Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System</p> - Table

Comparison of RMCE-in HEK and Flp-in HEK cells.

<p><b>(A)</b> Fluorescent and bright field pictures (BW) of RMCE-in HEK cells and commercial Flp-in^™293 cells transfected with the RMCE-in donor (top row) or the Flp-in donor (bottom row) acquired at day 2 (transient) and day 16 (stable) post-transfection. Emission acquired under 1s exposure and 100X magnification with GFP and RFP overlay. RFP⁺ RMCE-in HEK cells with transient GFP established a GFP⁺/RFP^- colony at day 16 (HygR selection). With the RMCE-in HEK cells, the Flp-in donor emits more GFP than the RMCE-in donor. No GFP is observed when the RMCE-in donor is used in the commercial Flp-in HEK cell line. (<b>B)</b> Comparison of RMCE-in and Flp-in systems ability to form colonies using either RMCE or Flp donor. Colony count of three individual experiment with ± SD. (<b>C-F)</b> PCR-validated gene shift in Flp-in (C), RMCE-in (D) HEK5.3, HeLa2.7, and mES II-F6 (duplex load) cells using the RMCE-in donor. Flp-in and HeLa2.7 show gene-shift in all sub-clones, HEK5.3 and mES II-F6 show 7/9 and 5/7 sub-clones with gene shift, as verified with Sanger sequencing. RMCE-in HEK5.3 and mES II-F6 sub-clone-absent PCR product indicates an incorrect gene shift. (<b>G)</b> <i>Top</i>: Schematic representation of incomplete RMCE. Integration into the FRT, site but not subsequent excision at the two F3 sites results in a scenario similar to that seen in Flp-in. CAG expresses HygR, but no RFP, generating resistance-RFP-negative clones. The false negative sub-clones are revealed by amplifying the region from AmpR to RFP in the RMCE-in docking site (represented by black arrows). <i>Bottom</i>: Positive PCR product from sub-clone four and six (HEK5.3 with the RMCE-in donor) is indeed, an example of such a scenario.</p

Design of the RMCE docking site.

<p>(A) <i>Left</i>: Schematics of the commercially available Flp-in docking site. <i>Right</i>: Design of the SB transposon constituting the RMCE docking site present in the new RMCE-in cell lines. The RMCE docking site contains the CAG promoter which drives the expression of a RFP reporter linked to the puromycin-resistant gene through the ribosomal skip element E2A. (B) Schematic representation of donor plasmid used for Flp-in (left) and RMCE-in (right). The genetic element of interest (GEI) is represented by a GFP reporter. Note that the GFP in the RMCE-in donor does not contain a poly(A)-signal and utilizes the poly(A) from the RMCE docking site. The RMCE-in and the Flp-in donor plasmid are compatible with both the RMCE-in and Flp-in cell line. (C) Post recombination of the Flp-in system <i>left</i>: Prokaryotic elements, the initial marker and selection gene are present in the commercial Flp-in^™-293 cells post recombination, while the RMCE-in <i>right</i> leaves no prokaryotic DNA or initial reporter genes after cassette exchange.</p

Validating flow cytometry results by fluorescence microscopy in RMCE-in HEK clones.

<p>HEK clones were exposed to 100X magnification and 1s exposure time for the assessment of RFP and GFP emission. All clones including colony 4.2 were RFP-positive and GFP with correlation to the high MFI value observed in flow cytometry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161471#pone.0161471.g003" target="_blank">Fig 3A</a>).</p