Bioreactor cultivation of CHO DP-12 cells under sodium butyrate treatment – comparative transcriptome analysis with CHO cDNA microarrays
© Klausing et al; licensee BioMed Central Ltd. 2011
Published: 22 November 2011
Sodium butyrate (NaBu) is not only known to inhibit proliferation but also to increase the specific productivity in cultivation of Chinese hamster ovary (CHO) cells  – the most commonly used mammalian cell line for pharmaceutical protein production . So far, little is known about the underlying mechanisms and genes that are affected by butyrate treatment. Besides the proteomic approach to unravel proteins involved in the processes, the analysis of transcriptomes presents another promising method. Here we show an application of our CHO cDNA microarray to identify genes associated with increased productivity during cultivation of CHO cells under sodium butyrate treatment.
Materials and methods
Four batch cultivations of CHO DP-12 cells (clone # 1934, ATCC CRL-12445) were performed in 2 L bioreactor systems under pO2- and pH-controlled conditions. In the exponential growth phase, 67 hours after inoculation, 2 mM sodium butyrate was added to three processes. The fourth was left untreated to function as control culture. Samples were taken before and then repeatedly after the addition of butyrate. RNA was isolated from cell pellets of 5·106 cells using TRIzol® Reagent (Invitrogen). For subsequent cDNA labeling, the Agilent Low-Input QuickAmp Labeling Kit (Agilent Technologies) was used. The custom designed 2 x 105 k cDNA microarray (Agilent Technologies) was spotted with 94,580 probes designed from CHO cDNA sequenced in-house. 38,310 of 41,039 sequenced contigs were used for the microarray, each covered by 2-4 probes . Data analysis was done with ArrayLims, EMMA, and SAMS, three CeBiTec based software tools . The raw data gathered by the microarray experiments were processed by standard Agilent background normalization and subsequent lowess normalization.
Fold change of selected genes from microarray analysis.
KEGG pathway category
Mean fold change in NaBu culture compared to control culture
# of probes
Transcription factor CA150b
Transcription & Translation
Coiled-coil domain containing 12
RNA-binding protein 8A
Protein kinase, membrane associated tyrosine/threonine 1
Myc proto-oncogene protein
Cdkn2c cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4)
Cyclin D1 (Ccnd1)
Programmed cell death 4
Phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1
Microarray analysis revealed a high number of regulated genes under sodium butyrate treatment in pathways like carbohydrate metabolism, cell cycle and signal transduction. Some of the regulated genes are promising targets for overexpression or knockdown/knockout experiments and we will further investigate the knockdown effect of selected genes using a siRNA approach in CHO cells. Our in-house microarray is suitable for further transcriptomic analysis of CHO cells under various conditions.
We would like to thank E. Schulte-Berndt (Institute for Genome Research and Systems Biology, CeBiTec, Bielefeld) for help with microarray hybridization and O. Rupp (Bioinformatics Resource Facility, CeBiTec, Bielefeld) for help with microarray data analysis.
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