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Title: Transcriptome analysis of CHO cells throughout fed-batch culture
Author: Longster, Joanne
ISNI:       0000 0004 5990 0900
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2015
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Chinese hamster ovary (CHO) cells are the predominant hosts for the production of recombinant protein. Although many engineering strategies have been employed to improve productivity, there is still a requirement to improve the process further and reduce costs of the therapies the proteins provide. This study profiles the expression of transcription factors (TFs), a group of cellular proteins that regulate gene expression, in order to identify novel engineering strategies. Two high-throughput transcriptome profiling techniques were used; Illumina Hi-seq 2000 RNA-sequencing and Affymetrix CHO gene 2.0 ST arrays. The techniques were compared for their attributes in profiling the CHO cell transcriptome throughout fed-batch culture. A reduction in the cost of transcriptome analyses, and their value in profiling proprietary industrial cell lines, means that they are likely to be carried out more often and therefore it will be valuable to know which technique would be most appropriate to use. There was a high significant correlation in expression and fold change of common and validated transcripts between the two datasets. However, additional filter steps were required to analyse the array data, as several of the probe-sets were non-unique or non-annotated. Furthermore, RNA-sequencing demonstrated a higher dynamic range and standard deviation, suggesting it may be more accurate at measuring low-expression transcripts and isoforms. The transcriptomic data was also used to profile the global gene expression trends that control the changes in cell physiology throughout culture. Functional analysis of differentially expressed transcripts demonstrated that between early stages of culture, the most regulated functional categories were metabolism and signal transduction, whereas in later stages of culture, the most regulated categories were cell cycle, DNA repair, DNA replication, and gene expression control. Through cluster analysis, cell cycle control was shown to be regulated in a relatively independent pattern to other functions, and was primarily down-regulated in later stages of culture. A novel method was also developed for identifying regulatory elements for use in novel synthetic promoters, based on the expression data throughout culture. This represents a valuable tool for developing constitutive and phase-specific gene expression systems in the future. Finally, the data was used to profile transcription factor expression and transcriptional regulation throughout culture. Transcription factors demonstrated expression ranges almost equal to those of the global dataset. However differentially expressed transcripts showed reduced fold changes in expression relative to the global dataset. Differentially expressed transcription factors, which were validated by both transcriptomic datasets, were identified, and those that could potentially be important for engineering strategies were highlighted.
Supervisor: James, D. C. ; Young, R. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available