Dynamic mRNA and miRNA profiling of CHO‐K1 suspension cell cultures

Abstract

In spite of the importance of Chinese hamster ovary (CHO) cells for recombinant protein production, very little is known about the molecular and gene regulatory mechanisms that control cellular phenotypes such as enhanced growth under serum-free conditions or high productivity. Most microarray analyses to this purpose are performed with samples taken during the exponential growth phase. However, the cellular transcriptome is dynamic, changing in response to external and internal stimuli and thus reflecting the current functional capacity of cells as well as their ability to adapt to a changing environment. Therefore, during batch or fed-batch cultivations it can be expected that the transcription pattern of genes will change and that such changes may give indications on the cellular state in terms of viability, growth, and productivity. In the current study we monitored the change in expression patterns of mRNAs and microRNAs (miRNA) during lag, exponential, and stationary phases in CHO-K1 suspension cell cultures. In total, over 1400 mRNAs and more than 100 miRNAs were differentially regulated (p<0.05) relative to the batch culture at the starting point. Functional clustering revealed groups of genes with similar expression patterns, which were subjected to functional pathway analysis. In addition, as miRNAs generally act as negative post-transcriptional regulators of mRNAs, we looked for changes in their expression that were inverse to those of their predicted target mRNAs.

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

“…After combination of equal amounts of cRNA of samples to be compared and fragmentation of the mixture, samples were hybridized to Agilent custom mouse 22k oligonucleotide microarrays (G4121B) as previously described [37,38]. In all arrays, a sample of the respective parental cell line was hybridized competitively in comparison with one of its subclones.…”

“…Quantitative real-time PCR of six individual genes was performed as described [37] examining RNA of all four subclones with primers (Supporting information, Table S1) designed for CHO mRNA [39] to confirm the microarray results (Supporting information, Fig. S1).…”

“…Data analysis was conducted as previously described [37,48,49] using the Genepix 4.1. for feature extraction and the LIMMA package under R/Bioconductor. Background correction of raw data was performed using the normexp function and intra-array normalization was performed using global lowess method.…”

Towards dynamic metabolic flux analysis in CHO cell cultures

“…In addition, large bioreactors of 10 000 L or more are common these days to satisfy the growing worldwide needs for therapeutic proteins [2,4,5]. Despite the intense research on CHO cells and related mammalian production cell lines, relatively little is known about intracellular metabolism of mammalian cells in cell culture [6]. This limited knowledge on metabolic fluxes and regulation of in vivo metabolism under industrially relevant culture conditions limits the potential of applying modern techniques such as metabolic engineering to further improve product yield, product quality and the overall process performance.…”

Editorial: Enhancing productivity of CHO, plant and algal cells

“…Juan A. Hernández Bort et al [2] present microarray data of CHO cells within different growth phases and are able to create a dynamic mRNA-miRNA network. Doolan et al [3] identify regulated genes in CHO cell fed-batch cultures, which showed either a decrease or a sustained cellular productivity.…”