To enable exploitation of a favorable chromosomal integration site, an initial primary (preliminary) genomic modification
to first mark or tag the site must be performed (Figure 1). This, in turn, creates a genomic platform that supports subsequent
modifications of that particular site. Although the first generation of tag-and-targeting approaches (so-called flip-in approaches) has limitations with respect to efficiency, RMCE is considered the method of choice for targeted integration mainly
because of the lack of reversibility, the lack of insertion of bacterial sequences, and the elimination of the tagging cassette
(as summarized in Gama-Norton, et al.).6
Figure 1. The recombinase mediated cassette exchange (RMCE) principle and strategy for efficient selection of correctly targeted
subclones. A single copy of a tagging cassette is initially integrated into the genome of the cell of choice through random
integration or viral mediated delivery of the DNA sequence (the tagging step). A reporter gene can be used to identify tagged
cell clones with desired expression levels. The cassette exchange-targeting step through RMCE is performed by co-transfecting
an Flp-encoding vector and a targeting vector containing the gene of interest (GOI). The latter is flanked by two heterospecific
Flp recognition target sites (FRT) that are identical to those flanking the tagging cassette. Additionally, the vector contains
an ATG start codon to restore the nonfunctional Δmarker-gene of the tagging cassette. Once the Flp enzyme has exchanged the
tagging for the targeting cassette, the ATG is placed in frame with the marker gene and cells can easily be selected by the
addition of the respective marker drug.10 Importantly, because of the incompatibility of the two heterospecific FRT sites,
excision reaction of the tagging cassette is excluded.
By tagging the genomic locus of interest, the heterotypic and incompatible recognition targets such as the Flp recognition
target (FRT) and FRTmut are introduced into a genomic locus (Figure 1).6,7 This creates a cassette acceptor allele, i.e., this tagged locus can now be used to integrate and exchange different DNA
cassettes of choice.
To target the genomic locus, a targeting vector containing the desired transgene flanked by the same set of heterotypic recognition
target sites as the tagged cassette is transfected together with an expression plasmid that encodes for the recombinase. A
double reciprocal crossover recombination event will lead to the exchange of the reporter cassette with the cassette of interest,
the targeting cassette. This strategy can hence be termed the tag and target strategy.8
Screening at the tagging stage (step 1) makes it possible to identify the integration sites that provide the desired expression
properties, i.e., high, stable, or regulated expression of the transgene. A successful selection process might require screening
large numbers of cells. Once this cell line is established, it will be further characterized for stability and safety. Further,
the optimal culture conditions that support maximal productivity will be defined. In this state, these cells can constitute
a platform that can be exploited to predictably express other proteins in a short time. This is illustrated in Figure 2.
Figure 2. Timelines to generate tagged high expression clones and constitute a producer cell line after targeting with the
gene of interest. Standard protocols to generate conventional producer cell lines rely on the establishment of a different
cell line for the expression of a different product. Commonly, more than 24 months are needed for the development and full
characterization of a biotechnologically relevant cell line. In contrast, the establishment of different cell lines that express
different products with biotechnological value by the application of RMCE technology can be achieved one-month after targeting
of an already tagged cell clone. This represents a tremendous advantage and opens up the possibility of creating a panel of
cell lines that express different genes of interest in a predictive manner.
The examples given below illustrate the performance of this technology for the establishment and exploitation of biopharmaceutical
producer cell lines.