Subphysiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells

Abstract

RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes; however, alternative messenger RNA (mRNA) splicing, has thus far, received little attention. In this study, we utilized RNASeq for transcriptomic analysis of a monoclonal antibody (mAb) producing CHO K1 cell line subjected to a temperature shift. More than 2,465 instances of differential splicing were observed 24 hr after the reduction of cell culture temperature. A total of 1,197 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using quantitative polymerase chain reaction in the mAb‐producing CHO K1 cell line used for RNASeq and a further two CHO K1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of these changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilized to gain a deeper understanding of post‐transcriptional regulation in CHO cells during biopharmaceutical production.

Expanding the Chinese hamster ovary cell long noncoding RNA transcriptome using RNASeq

“…Next, we compared the 389 differentially expressed lncRNAs to the 1,312 differentially expressed protein-coding genes identified from the same data set a similar count-based gene-level analysis (Tzani et al, 2020). Twenty-three differentially expressed proteincoding genes were found to have to have either an overlapping antisense lncRNA or a divergent lncRNA upstream that was also differentially expressed.…”

“…The RNASeq data utilised for this analysis was generated as part of a study in our laboratory to advance our understanding of the transcriptomic response to decreasing CHO cell culture temperature (i.e., "temperature shift"; Tzani et al, 2020). Briefly, the experimental design of the study involved growing 8 biological replicates of a monoclonal antibody (mAb) producing CHO K1 cell line for 48 hr at 37°C.…”

“…The temperature of four replicates was then reduced to 31°C while the remaining four replicates were maintained at 37°C before all samples were submitted for transcriptomic analysis at 24 hr posttemperature shift. The transition to subphysiological cell culture temperature resulted in a reduction of growth rate, alteration of extracellular metabolite concentration as well as widespread changes in mRNA abundance and splicing (Tzani et al, 2020).…”

“…While much work remains, this study is an important step towards unlocking the potential of lncRNAs for rational genetic engineering of CHO cell lines. 2 | MATERIALS AND METHODS 2.1 | Cell culture, library preparation, and RNA sequencingThe cell culture protocol, profiling of extracellular metabolites, library preparation and RNA sequencing are described inTzani et al (2020).…”

Tracing production instability in a clonally-derived CHO cell line using single cell transcriptomics

“…Next generation sequencing (NGS) has enabled the acquisition of multiple high-quality publicly available reference genomes (Kelly et al, 2017;Lewis et al, 2013;Rupp et al, 2018;Xu et al, 2011), which has in turn, facilitated the analysis of new features of CHO cell biology including DNA methylation (Wippermann, Rupp, Brinkrolf, Hoffrogge, & Noll, 2017) and epigenetics (Feichtinger et al, 2016) as well as improving mass spectrometry-based proteomics (Meleady et al, 2012). RNA-seq has also been utilised to characterise changes not only in gene expression (Clarke et al, 2019;Sha, Bhatia, & Yoon, 2018) but also alternative splicing of CHO cell mRNAs (Tzani et al, 2020). The technology has also played a crucial role in the annotation of noncoding RNA molecules such as microRNAs (Hackl et al, 2012) and long non-coding RNAs (Hernandez et al, 2019;Motheramgari et al, 2020;Vito et al, 2020).…”