Cre/Lox-based RMCE for efficient GOI exchange in CHO cells
Cre recombinase allows double recombination events between two pairs of
heterologous Lox sites, allowing LP to be exchanged with TV
(Saraf-Levy et al., 2006). To perform
Cre/Lox-mediated RMCE, 2 μg of TV DNA was co-transfected with 2 μg of
Cre expression vector in clones targeted by LP (Fig. 4A). Since TV has
no promoter, GFP will be expressed only when LP is correctly exchanged
with TV (Fig. 4A). Therefore, in order to select the successfully
exchanged clones, cells expressing GFP were identified during the single
cell cloning procedure. All established clones expressed GFP but not
mCherry, indicating RMCE-mediated mCherry-to-GFP swapping (Fig.4B).
We then examined the nucleotide sequence of the swapped region. PCR was
performed on genomic DNA with primer sets before and after Lox5171 (Fig.
4C). A band corresponding to the expected size appeared in GFP-positive
clones obtained after RMCE, whereas the PCR band did not appear in
clones obtained before RMCE (Fig. 4C; red arrow). Subsequent DNA
sequencing of the purified PCR product revealed that the sequence was
identical to that of the mCherry-to-GFP swapped Fer1L4 region
(Fig. 4C).
The success of the Cre/Lox-based RMCE system allowed us to investigate
the mCherry-to-GFP swap rate by evaluating the percentage of cells
expressing only GFP, without expressing mCherry (Fig. 4D). Before RMCE,
the percentage of such cells was 0.5%, indicating that a small
proportion of clones targeted by LP exhibit autofluorescence that can be
detected in the GFP filter (Fig. 4D). After RMCE, the percentage of
these cells significantly increased to 2.95%, indicating that the
Cre/Lox-based RMCE system successfully functioned in CHO cells (Fig.
4D).