Browsing by Subject "Baby hamster kidney cell"

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    High-throughput transcriptomic analysis of resource-poor mammalian cell lines for recombinant protein production
    (2013-10) Johnson, Kathryn Christine
    Over the past 30 years, mammalian cell culture has enabled the production of recombinant protein therapeutics for treatment of a broad range of debilitating or life-threatening diseases. Continual improvements in cell and process engineering have facilitated the attainment of once unheard-of product titers, and improvements in molecular analysis techniques and process analytic technologies have been employed with great success for cell culture process characterization. High-throughput transcriptomic analysis tools such as microarrays and next-generation RNA sequencing (RNA-seq) provide access to gene expression information by simultaneously measuring the expression levels of tens of thousands of genes. However, until recently such tools have not been used to their full advantage in mammalian cell culture processes due to limitations in available reference sequences for the industrially important Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cell lines. We employed high-throughput RNA sequencing in several CHO cell lines to identify and interrogate a class of small non-coding RNAs called microRNAs (miRNAs), which mediate post-transcriptional repression of protein-coding genes. We annotated and analyzed the expression and genomic conservation of several hundred of these small RNAs. We also employed RNA sequencing to build a comprehensive reference transcriptome for a recombinant protein-producing BHK cell lines. We utilized the BHK reference sequence to enable analysis of gene expression levels in the BHK cell line and two Syrian hamster tissues. We designed an expression microarray from the BHK sequence and utilized it to analyze the transcriptome profiles of BHK cells at several time points in perfusion culture at manufacturing scale. Implementation of several functional analysis tools revealed a consistent time-dependent change in the transcriptome profile that involved down-regulation of extracellular matrix components and changes to calcium signaling genes. The transcriptomic reference sequences we developed in this research and the detailed studies they have enabled will enhance our ability to understand and further optimize cell culture processes.