doi: 10.1371/journal.pone.0161471.
eCollection 2016.
Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System
Affiliations
- PMID: 27541869
- PMCID: PMC4991790
- DOI: 10.1371/journal.pone.0161471
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Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System
PLoS One.
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Abstract
Recombinase mediated cassette exchange (RMCE) is a powerful tool for targeted insertion of transgenes. Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increased efficiency of integration of the genetic element of interest (GEI) at a single docking site, lack of bacterial backbone at the docking site both before and after GEI integration, removal of selection and visual markers initially present at the docking site upon GEI integration and the possibility to validate GEI integration by loss of a red fluorescence reporter. Moreover, the RMCE-in cell lines are compatible with GEI donors used for the Flp-in system. We demonstrate a three-step procedure for generating RMCE-in cell lines, (I) RMCE-in transposon and SB10 transposase transfection, (II) clone isolation, and (III) selecting single integrated clones with highest RFP level, which could in principle be used to turn any cell line into an RMCE-in cell line. The RMCE-in system was used as a proof of concept to produce three new RMCE-in cell lines using HEK293, HeLa, and murine embryonic stem (mES) cells. The established RMCE-in cell lines and vector are freely available from the ATCC cell bank and Addgene respectively.
Conflict of interest statement
Figures
(A) Left: Schematics of the commercially available Flp-in docking site. Right: 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 left: Prokaryotic elements, the initial marker and selection gene are present in the commercial Flp-in™-293 cells post recombination, while the RMCE-in right leaves no prokaryotic DNA or initial reporter genes after cassette exchange.
(A) Southern blot of selected genomic DNA from KpnI digested HEK, (B) HeLa and PstI digested (C) mES clones. A 700bp dCTP32 labeled RFP probe (red rectangle Fig 1A) identified transposition of the gene cassette at bands > 2900bp as illustrated in Fig 1A. Clone numbers are depicted above each lane.
(A) Representative flow cytometry results by RMCE-in from the HEK (B) HeLa (C) and mES clones. RFP emission compared with WT cells (shown in gray). All HEK, mES clones, and HeLa clone 2.7 showed a single RFP peak indicating homogenous populations. Right: MFI values of RMCE-in HEK, HeLa, and mES cell clones (Clone numbers correspond to the Southern blot shown in Fig 2) are shown. Cell singlet mean [min-max] counts; RMCE HEK clones 8829[3194–11875] HeLa clones 15369[5588–21004], mES clones 49757[42637–57742]
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 (Fig 3A).
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 (A), and mES colony II-F1, II-H2, and I-F10 (B). Plasmid backbone (grey) and 5’ LIR sequences (purple) obtained by Sanger sequencing are shown above the area of insertion.
(A) 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) 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. (C-F) 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. (G)
Top: 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). Bottom: Positive PCR product from sub-clone four and six (HEK5.3 with the RMCE-in donor) is indeed, an example of such a scenario.
(A) Representative flow cytometry results of Flp-in™ sub-clone 1 using the HEK Flp-in system testing the RMCE (upper) and Flp (lower) donor compared with WT HEK (grey). (B) The RMCE (upper) and Flp (lower) sub-clones RFP and GFP MFI with SD. Cell singlet mean [min-max] counts; Flp sub-clones 20988[16977–23555], RMCE sub-clones 20002[16865–23680]. (C) The copy number normalized to GLIS6 of either Flp (red) or RMCE (blue) gene-shifted sub-clones. Non-parametric bootstrap for confidence limits (upper and lower bar) for the populations mean (middle bar). (D) Representative flow cytometry results of RMCE-in HEK sub-clone 1 using the HEK RMCE-in system testing the RMCE (upper) and Flp (lower) donor compared with HEK5.3 (grey). (E) The RMCE (upper) and Flp (lower) sub-clones RFP and GFP MFI with SD. Cell singlet mean [min-max] counts; Flp sub-clones 15312[104–19093], RMCE sub-clones 20168[16867–24292]. (F) The copy number normalized to GLIS6 of either Flp (red) or RMCE (blue) gene-shifted sub-clones. Non-parametric bootstrap for confidence limits (upper and lower bar) for the populations mean (middle bar)