Establishment of optimal conditions for RMCE
The ratio between TV and Cre recombinase is a decisive factor in RMCE (Du, Hu, Ayala, Sauer, & Zhang, 2009). Thus, to improve RMCE efficiency, we investigated the optimal ratio between TV and Cre recombinase. To achieve this, 2 μg of TV DNA was co-transfected with various amounts of Cre expression vector in clones targeted by LP. Further, 2 μg TV with 0.5 μg Cre expression vector significantly increased the percentage of cells expressing GFP compared to the initial conditions (2 μg TV DNA with 2 μg Cre expression vector) (Fig. 5A). These results indicated that a 4:1 ratio involving TV (2 μg) and Cre expression vector (0.5 μg) was the optimal condition for RMCE (Fig. 5A).
The application of multi-cistronic gene cassettes capable of simultaneously expressing GOI and GFP in TV may be a breakthrough for RMCE; GOI can be used for protein production, and GFP can be used as a sorting marker for selecting swapped clones (Bouabe, Fässler, & Heesemann, 2008). Thus, we performed FACS sorting to enrich GFP-positive clones established after the identification of the optimal RMCE conditions. FACS sorting enhanced the percentage of GFP-positive cells from 3.94% up to 80%, indicating that RMCE efficiency can be indirectly increased by enriching GFP-positive cells capable of simultaneously expressing GOI (Fig. 5B).