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.

Synthetic biology in multicellular organisms: Opportunities in nematodes

“…Recombinase‐mediated cassette exchange has been widely used in mammalian cell culture (Callesen et al, 2016) and was adapted to insert DNA sequences at specific sites in the genome of Drosophila melanogaster (Bateman et al, 2006; Callesen et al, 2016). Recently, Nonet (2020) extended this approach to C. elegans with constitutively expressed fluorescent reporters as well as bipartite expression systems (discussed in Section 4.1).…”

Nonmonotone invasion landscape by engineered control of metastasis activator levels

“…1B), we used recombinase-medicated cassette exchange (RMCE) to integrate a mammalian negative-feedback (mNF) synthetic gene circuit into one LP clone (MB231-1 and 293-3) from each cell type. RMCE [71][72][73] relies on Flp 74 and other recombinases to repeatedly replace DNA in a predefined LP without double strand breaks in various mammalian cell lines 43,62,63 . The mNF gene circuits 75 (Fig.…”

λ-integrase mediated seamless vector transgenesis platform

“…One of the best examples of Cre-lox recombination system application is the "Brainbow" that enable different color labelling of each neuron of mice to create a detailed map of the neuronal network allowing neuroscientists to study neurons and glial cells as a function of time (158). The Cre/loxP and Flp/FRT systems are widely used in recombination-mediated cassette exchange (RMCE) strategy to engineer the genome of mammalian cell lines for the production of monoclonal antibodies (159,160). However, transgenesis of exogenous DNA is inefficiently catalyzed by these recombinases because they can predominantly catalyze the excision reaction in an in vivo condition leading to excision of integrated DNA from the genome.…”