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

Morten M Callesen,Annette C Füchtbauer,Ernst-Martin Füchtbauer,Martin F Berthelsen,Jannik E Jakobsen,Sira Lund,Peter Hohenstein

doi:10.1371/journal.pone.0161471

Morten M Callesen, Annette C Füchtbauer + Show 5 more

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https://doi.org/10.1371/journal.pone.0161471

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Journal: PloS one	Publication Date: Aug 19, 2016
Citations: 3	License type: CC BY 4.0

Affiliation: Aarhus University

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.

Highlights

Functional investigation of genetic elements such as promoters, protein coding genes, non-coding RNA, etc. in a transgenic gain-of-function approach is often hampered by the regulatory influences of sequences flanking the transgene integration site
The murine embryonic stem (mES) cell clones were produced by electroporation of linearized Recombinase mediated cassette exchange (RMCE)-in transposon plasmid cleaved outside the left and right inverted repeats (LIR, RIR), to increase the likelihood of single-integrations
The loss of red fluorescence protein (RFP) upon targeted genetic element of interest (GEI) integration, which is possible in our RMCE system was used to indicate a successful exchange of the transgene

Summary

Introduction

Functional investigation of genetic elements such as promoters, protein coding genes, non-coding RNA, etc. in a transgenic gain-of-function approach is often hampered by the regulatory influences of sequences flanking the transgene integration site. In a transgenic gain-of-function approach is often hampered by the regulatory influences of sequences flanking the transgene integration site. This so-called positional effect can be circumvented by placing the genetic element of interest (GEI) in a pre-determined, fixed position in the genome. The Flp-in system succumb to a genuine problem by co-integrating several prokaryotic elements of the plasmid backbone with insertion of the GEI. In addition to the plasmid backbone, the initial lacZ-Zeocin selection marker gene resides in the docking site with reported read-through by the Flp-in protocol [6]. It has been reported that the Flp-in system does not select against expression from additional, randomly integrated GEI[3,8], which may interfere and obstruct the experiments

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