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

Abstract

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