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When expressing pharmaceutical recombinant proteins in mammalian cells, the protein is commonly directed through the secretory pathway, in a signal peptide-dependent manner, to acquire specific post-translational modifications and to facilitate secretion into the culture medium. One key premise for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding the therapeutic proteins, erythropoietin (EPO) and Rituximab, was determined. The results show that ER-directed recombinant mRNAs exhibited an efficient recruitment to the ER when compared to an endogenous ER-directed mRNA, with no cytoplasmic translation of ER-directed recombinant proteins observed. These observations indicate that the recombinant mRNA, encoding ER-directed proteins, follows the same distribution pattern as endogenous mRNA directed towards the ER. Furthermore, the previous established fractionation method proves to be an efficient tool to study not only recombinant mRNA localization, but also recombinant protein trafficking between the ER and cytosol in CHO cells.

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9-expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off-target effects were observed from off-target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene-disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild-type CHO-S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.

Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification of EPO in a high-throughput setting.

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.

In S. cerevisiae cells debilitated in mRNA nuclear export, transcripts are retained in nuclear foci ("dots"). The ultimate fate of dot-mRNA has remained elusive. Here, we use single molecule counting microscopy and (35)S-methionine pulse-labeling assays to quantify cytoplasmic HSP104 RNA levels and estimate HSP104 RNA translation status. HSP104 transcripts, retained in dots as a consequence of the mex67-5 mutation, are slowly released over time for cytoplasmic translation. Thus, dot-mRNA retains function. However, forcing its nuclear export, by overexpressing the Sub2p mRNA export factor, does not elevate Hsp104p protein levels but is instead paralleled by growth deficiency. Nuclear export and growth phenotypes are both counteracted by coexpressing the nuclear RNA quality control factor Rrp6p. Thus, prematurely released dot-mRNA is translationally inactive and possibly toxic. Accordingly, nuclear retention of mRNA may serve a precautionary role during stressful situations such as, e.g., decreased mRNA maturation competence.