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).