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.