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).