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Revisiting the impact of C12orf35 locus as a genomic hot spot and engineering target for cell line development using recombinase-mediated cassette exchange

  • 조규희 (Kyuhee Cho, 아주대학교 일반대학원)

초록/요약

Traditional Chinese hamster ovary (CHO) cell line development is based on random integration of transgene that causes clonal variation and subsequent large- scale clone screening. Therefore, site-specific integration (SSI) of transgenes into genomic hot spots has recently emerged as an alternative method for cell line development. However, the specific mechanisms underlying hot spot site formation remain unclear. In this study, we aimed to generate landing pad cell lines via the random integration of transgenes encoding fluorescent reporter proteins flanked by recombination sites to facilitate recombinase-mediated cassette exchange. The random integration-based landing pad cell line expressing high reporter levels with spontaneous C12orf35 locus deletion exhibited similar reporter fluorescent protein levels compared to targeted integrants with an identical reporter landing pad construct at the CHO genome hot spot, the C12orf35 locus. Additionally, Resf1, a C12orf35 locus gene, knockout in the random integration-based landing pad cell line with conserved C12orf35 increased reporter expression levels, comparable to those in cell lines with C12orf35 locus disruption. These results indicate that the effect of SSI into the C12orf35 locus, a genomic hot spot, on high-level transgene expression was caused by C12orf35 disruption. In contrast to C12orf35 knockout, knockout at other well-known hot spot sites at specific loci of genes, including Fer1L4, Hprt1, Adgrl4, Clcc1, Dop1b, and Ddc, did not increase transgene expression. Overall, our findings suggest that C12orf35 is a promising engineering target and a hot spot for SSI-based cell line development. In addition to the hot spot editing, to overcome the production capacity limitations of RMCE master cell lines used for antibody production, the potential problem of fluorescence-based cell line screening, where differences in fluorescence intensity do not correspond to those in antibody productivity, must be addressed. Therefore, we propose an applicable follow-up strategy to reduce fluorescence intensity through PEST motif and ubiquitin-dependent proteolysis. Keywords: Cell line development; RMCE; Hot spot; C12orf35; Knockout; Protein degradation; Fluorescence-based screening

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목차

1. Introduction 1
2. Materials and Methods 3
2.1 Cloning of sgRNA/Cas9 vector and donor plasmid 3
2.2. Cell line and culture maintenance 4
2.3. Generation of LP RI cell pools and single cell derived cell lines 4
2.4. Generation of recombinant antibody producing cell lines 5
2.5. Generation of LP targeted integration cell pools and single cell derived cell lines 5
2.6. Validation and generation of C12orf35 KO clones and KO cell pools 5
2.7. Restoration of EGFP LP RI cell lines from recombinant antibody producing cell lines 6
2.8. Flow cytometry analysis 7
2.9. Batch culture. 7
2.10. Quantitative real-time PCR 8
2.11. Statistics analyses 8
3. Results & Discussion 25
3.1. Establishment and characterization of high-EGFP expressing RMCE MCLs for monoclonal antibody production 25
3.2. Comparison of productivity between RI-based and C12orf35 hot spot-targeting RMCE MCLs 30
3.3. Impact of Resf1 knockout on EGFP expression in LP RI C4, C12orf35 conserved LP RI single cell clones. 34
3.4. Exploration of hot spot sites and the specific role of Resf1 disruption in enhancing transgene expression 37
4. Perspective & Conclusion 44
5. References 47

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