Efficient Flp-Int HK022 dual RMCE in mammalian cells

Abstract

Recombinase-mediated cassette exchange, or RMCE, is a clean approach of gene delivery into a desired chromosomal location, as it is able to insert only the required sequences, leaving behind the unwanted ones. RMCE can be mediated by a single site-specific DNA recombinase or by two recombinases with different target specificities (dual RMCE). Recently, using the Flp–Cre recombinase pair, dual RMCE proved to be efficient, provided the relative ratio of the enzymes during the reaction is optimal. In the present report, we analyzed how the efficiency of dual RMCE mediated by the Flp–Int (HK022) pair depends on the variable input of the recombinases—the amount of the recombinase expression vectors added at transfection—and on the order of the addition of these vectors: sequential or simultaneous. We found that both in the sequential and the simultaneous modes, the efficiency of dual RMCE was critically dependent on the absolute and the relative concentrations of the Flp and Int expression vectors. Under optimal conditions, the efficiency of ‘simultaneous’ dual RMCE reached ∼12% of the transfected cells. Our results underline the importance of fine-tuning the reaction conditions for achieving the highest levels of dual RMCE.

Efficient Genome Manipulation by Variants of Site-Specific Recombinases R and TD

“…Both the integration and the replacement activity of the R and TD variants are comparable to that of Flp and HK022 [25, 26]. In fact, under optimal conditions the integration efficiency of TD-40 and R1-6 is higher than of any other tyrosine recombinase we experimented with: ~5–6% and ~1.5–3% of the transfected cells, respectively (Figs.…”

“…We next tested whether the TD variant can be paired with the R variants to perform the dual recombinase-mediated cassette exchange, dual RMCE [6, 10, 25, 26]. In these experiments we utilized the assay similar to that we used to analyze the replacement activity of the Flp/HK022 Int recombinase pair [26].…”

“…In these experiments we utilized the assay similar to that we used to analyze the replacement activity of the Flp/HK022 Int recombinase pair [26]. A schematic of this assay is shown in Fig.…”

“…4). Since these variants do not cross-react with FRT and loxP , and Flp and Cre do not react with R1RT or TDRT , the evolved variants of R and TD can be therefore paired with these dual RMCE-proved recombinases [6, 10, 25, 26] to mediate multifaceted replacement of genomic fragments.…”

“…3d, 5a). In contrast, the integration efficiency of HK022 Int is about 0.8% of transfected cells [26]. Flp, as a control, routinely gives us integration in ~0.1% of the transfected cells (as we did not optimize the parameters of this reaction, it is possible that Flp can mediate integration more efficiently).…”