Bioinformatic Identification of Chinese Hamster Ovary (CHO) Cold‐Shock Genes and Biological Evidence of their Cold‐Inducible Promoters

Abstract

Lower cultivation temperature dramatically affects cell growth and cellular productivity in Chinese hamster ovary cells (CHO) and is often used in industrial applications with the aim to enhance productivity. Cold‐inducible proteins whose activity is induced at lower temperature play an important role in understanding the mechanisms of cold‐induced changes in gene expression. One of these mechanisms is increased transcription of specific target genes controlled by sequence elements in cold‐inducible promoters. This study provides a list of cold‐inducible genes and endogenous cold‐inducible promoters of CHO cells. Transcriptome data before and after a temperature shift from 37 to 33 °C are analyzed to identify 94 cold‐inducible genes, which are highly expressed and have a high fold change in expression after the temperature shift. Cold‐inducible promoters are identified from the top ten cold‐inducible genes, showing up to 11‐fold increased luciferase expression at lowered temperature in transient transfections. Additionally, several common transcription factor binding sites are identified in the top cold‐inducible promoter sequences and expression of their corresponding transcription factors over temperature‐shift cultivation is evaluated. These may be responsible for enhanced promoter activity under lower temperature and can be used as engineering targets.

Temperature Down‐Shift Modifies Expression of UPR‐/ERAD‐Related Genes and Enhances Production of a Chimeric Fusion Protein in CHO Cells

“…The CMV promoter (which is one of the most used for the r‐protein expression and used in the current study) exhibited an increased promoter activity at low temperature and a fourfold and 1.7‐fold increase in transient and stable expression studies with r‐genes. [ 37 ] These observations are in the line with the r‐gene expression data presented in the current work (Figure 2C,D) and offers a molecular explanation why lowered culture temperature generally induces an increased expression of r‐genes. In conclusion, a lowered temperature contributes to a better secretory phenotype in CHO cells by modifying the expression of genes involved in the UPR and ERAD pathways.…”

“…Increased q hEPO‐Fc s in low temperature cultures also coincided with a higher amount of the r‐gene (Figure 2C,D) and arrested cell cycle in G1/G0 phase (Figure 1D), while improved cell viability did not contribute to better productivities as the q hEPO‐Fc s were higher at 32 °C that 37 °C in both batch and fed‐batch at timepoints when the viability was 100% in every condition. The amount of hEPO‐Fc reported here was in the range of previous studies (from 0.02 mg L −1[ 36 ] to 200 mg L −1[ 37 ] under different culture conditions). In addition, the higher q hEPO‐Fc and Y hEPO‐Fc in low temperature cultures were consistent with previous reported data, indicating an increased r‐protein production and cell‐specific productivity due to a decrease in culture temperature.…”

“…In previous publications, a temperature decrease in CHO cells increased the amounts of r‐gene mRNA by a mechanism associated with enhanced mRNA stability [ 20 ] as well via increased transcriptional activity. [ 37 ] In our studies we have not distinguished if changes to transcript amounts (r‐gene RNA or other targets) arises as a result of changes to mRNA production or stability in response to low temperature culture. This caveat applies to the mechanisms responsible for changes to all transcripts measured in this study.…”

Key Challenges in Designing CHO Chassis Platforms

“…With the recently emerged and still emerging tools, such model-based designed engineering approaches become more and more feasible, where multiple genes are targeted to achieve a specific purpose. New circuits and tools from synthetic biology [222][223][224], including as an example a panel of both endogenous promoters with defined properties [133,225] as well as artificial ones that can be adapted and modified as needed [226,227], will contribute and will be more and more required. Such engineering approaches will require a diverse set of such control units that are based on different mechanisms, as more and more genes may have to be controlled at different expression levels.…”

Enhancement of Transgene Expression by Mild Hypothermia Is Promoter Dependent in HEK293 Cells

“…However, the beneficial effect of MH cultivation on increased productivity has been reported to be cell line specific [ 6 , 7 , 8 ]. Such clonal variation in transgene expression can originate from several factors [ 9 ], and recent studies have unveiled mechanistic insights into differences in protein expression and cell culture performance under MH, which include transgene integration sites, vector elements, regulation of specific genes involved in carbon metabolism and unfolded protein response, and expression of cold-shock proteins, albeit these studies are mainly conducted in CHO cells [ 8 , 10 , 11 , 12 ]. In the context of stable cell line development, the choice of a promoter is crucial because promoter activity determines both transgene expression levels and expression patterns under specific culture environments [ 8 , 9 ].…”