Figure Legends
Fig. 1. Schematic of LP and TV. (A) LP was designed where Lox5171/LoxP
sites flanked mCherry, followed by a CMV promotor-driven thymidine
kinase (TK ) gene. LP also contained the 5′ HA (0.2 kb region ofFer1L4 exon 1 present upstream of the transcription start site)
and 3′ HA (0.2 kb region of Fer1L4 exon 1 present after the start
site). (B) TV was designed where Lox5171/LoxP sites flanked GFP,
followed by an SV40 promoter-driven neomycin resistance gene.
Fig. 2. Targeted integration of LP mediated by CRISPR/Cas9. (A)
Schematic diagram of CRISPR/Cas9-mediated SSI of LP in Fer1L4region. Only when RMCE LP was incorporated into Fer1L4 exon 1,
mCherry was expressed under the control of the endogenous Fer1L4promoter. (B) Differential interference contrast (DIC) and fluorescence
microscopy images of single cell clones targeted by LP (red: mCherry,
scale bar: 50 μm). (C) Schematic diagram: PCR reaction was performed to
determine Fer1L4 copy number targeted by LP. Fer1L4 Fwd
primer was designed to bind to the sequence before the 5′ HA, andFer1L4 Rev primer was designed to bind to the sequence after the
3′ HA. When Fer1L4 is not targeted by LP, 200 bp PCR product will
be amplified. However, when Fer1L4 is targeted by LP, 3,300 bp
PCR product will be amplified. (D) Comparison of relative Fer1L4copy number between a negative control and clones targeted by LP. To
amplify only the Fer1L4 locus not targeted by LP, qPCR was
performed with a 5 s extension time. Eight of 12 clones showed half the
value compared to the negative control, indicating that only one copy of
the Fer1L4 genomic region was targeted by LP (arrowhead).
Fig. 3. Verification of recombined sequences in single cell clones
targeted by LP. (A) Schematic diagram of nested PCR to amplify the
LP-targeted region. The 1st Fwd primer was designed to
bind to the sequence before the 5′ HA, and the 1st Rev
primer was designed to bind to the mCherry sequence. The
1st PCR amplification with 1stprimer set generated the anticipated amplicon (590 bp), which was used
as template for the 2nd PCR. Subsequently, the
2nd PCR amplification with 2ndprimer set generated the anticipated amplicon (436 bp), which was used
as templates for the 3rd PCR. (B) Picture of agarose
gel electrophoresis showing the PCR amplicon generated by the nested
PCR. The 3rd PCR amplification with the
3rd primer set generated an amplicon (348 bp) in clone
#11 (red arrow). (C) The DNA sequence of the amplified PCR product was
identical to that of the Fer1L4 region targeted by LP.
Fig. 4. Cre/Lox-based RMCE for efficient GOI exchange in CHO cells. (A)
Schematic diagram of GOI swapping using Cre/Lox-based RMCE. Cre
recombinase allows double recombination between two pairs of
heterologous Lox sites, exchanging LP with TV. Since TV has no promoter,
GFP will be expressed if LP is correctly exchanged with TV. (B) DIC and
fluorescence microscopy images of single cell clones generated by RMCE
(red: mCherry, green: GFP, scale bar: 50 μm). The success of these
Cre/Lox-based RMCE resulted in mCherry-to-GFP swapping, allowing the
expression of GFP rather than mCherry. (C) Schematic diagram of PCR to
amplify swapped regions. PCR was performed on genomic DNA with primer
sets targeting before and after Lox5171. A band corresponding to the
expected size appeared in GFP positive clones, whereas no PCR band
appeared in the negative control. The DNA sequence of the amplified PCR
product was identical to that of the mCherry-to-GFP swapping region inFer1L4 . (D) The mCherry-to-GFP swapping rate was measured by flow
cytometry. The percentage of cells expressing GFP without mCherry was
calculated before and after RMCE (**P < 0.01,
Mann-Whitney U test). Means ± S.D., N = 3.
Fig. 5. Establishment of optimal conditions for RMCE. (A) 2 μg of TV DNA
was co-transfected with various amounts of Cre expression vector in
clones targeted by LP. The percentage of cells expressing GFP, but not
mCherry, was measured by flow cytometry. (**P < 0.01,
Mann-Whitney U test). Means ± S.D., N = 3. (B) FACS
sorting-mediated enrichment of GFP-positive cells established under the
optimized RMCE conditions. The percentage of cells expressing GFP, but
not mCherry, was measured by flow cytometry. (**P <
0.01, Mann-Whitney U test). Means ± S.D., N = 3.
Fig. 6. Cre/Lox based RMCE reestablishes new clones that expect similar
productivity and properties compared to existing ones.
Fig. 7. Application of multi-cistronic gene cassettes capable of
simultaneously expressing GOI and GFP in TV. Multiple GOI linked to 2A
can be used for protein production and GFP can be used as a sorting
marker for selecting swapped clones.