title: Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System.
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.
authors.affiliation: Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus C, Denmark.|Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus N, Denmark.
authors.forename: Morten M
authors.identifier: http://orcid.org/0000-0001-6258-2171
authors.initials: MM
authors.lastname: Callesen
authors.affiliation: Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus C, Denmark.|Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus N, Denmark.
authors.forename: Martin F
authors.initials: MF
authors.lastname: Berthelsen
authors.affiliation: Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus C, Denmark.
authors.forename: Sira
authors.initials: S
authors.lastname: Lund
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.
authors.forename: Annette C
authors.initials: AC
authors.lastname: Füchtbauer
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.
authors.forename: Ernst-Martin
authors.initials: EM
authors.lastname: Füchtbauer
authors.affiliation: Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus C, Denmark.|Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus N, Denmark.
authors.forename: Jannik E
authors.initials: JE
authors.lastname: Jakobsen
mesh_terms: D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D018390:Gene Targeting; D017930:Genes, Reporter; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D045522:Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic
chemical_list: D045522:Recombinases; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase