Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering

Abstract

Chinese hamster ovary (CHO) cells are the predominant cell factory for the production of recombinant therapeutic proteins. Nevertheless, the lack in publicly available sequence information is severely limiting advances in CHO cell biology, including the exploration of microRNAs (miRNA) as tools for CHO cell characterization and engineering. In an effort to identify and annotate both conserved and novel CHO miRNAs in the absence of a Chinese hamster genome, we deep-sequenced small RNA fractions of 6 biotechnologically relevant cell lines and mapped the resulting reads to an artificial reference sequence consisting of all known miRNA hairpins. Read alignment patterns and read count ratios of 5′ and 3′ mature miRNAs were obtained and used for an independent classification into miR/miR* and 5p/3p miRNA pairs and discrimination of miRNAs from other non-coding RNAs, resulting in the annotation of 387 mature CHO miRNAs. The quantitative content of next-generation sequencing data was analyzed and confirmed using qPCR, to find that miRNAs are markers of cell status. Finally, cDNA sequencing of 26 validated targets of miR-17-92 suggests conserved functions for miRNAs in CHO cells, which together with the now publicly available sequence information sets the stage for developing novel RNAi tools for CHO cell engineering.

Annotation of additional evolutionary conserved microRNAs in CHO cells from updated genomic data

“…Next‐generation sequencing (NGS) data were obtained by Illumina sequencing as described by Hackl et al (2011) and is available at the Sequence Read Archive SRA (www.ncbi.nlm.nih.gov/sra/), accession number SRA024456.1. Cell lines sequenced included CHO‐DUXB11 (ATCC CRL‐9096), CHO‐K1 (ECACC‐CCL‐61), and two derivative recombinant cell lines producing a monoclonal antibody (CHO‐K1) and an EpoFc fusion protein (DUXB11).…”

“…In 2011, the first systematic approaches to miRNA annotation in CHO cells were reported (Hackl et al, 2011; Johnson et al, 2011). Subsequent studies were focused on providing insights into the importance of miRNAs for molecular pathways relevant to cell culture engineering, such as growth (Jadhav et al, 2014), apoptosis (Druz et al, 2011), and protein secretion (Barron et al, 2011; Loh et al, 2014).…”

“…For classification as novel cgr‐miRs, the next‐generation sequencing (NGS) read counts from new and existing datasets (Hackl et al, 2011) of these sequences were examined using more than five read counts as cut‐off for the existence of these miRNAs. Thereby, 383 sequences did not show expression under the constraints for valid NGS signals, leaving 71 NGS confirmed miRNAs.…”

Unveiling the principle of microRNA-mediated redundancy in cellular pathway regulation

“…RNA deep sequencing data of different CHO lines indicated that approximately 400 miRNAs are expressed in CHO cells. 48 Although Chinese hamsters possess a considerably lower number of chromosomes (n=22) compared to mice (n=40), the genomic information is highly overlapping. 49 Thus, the presence of only 400 different miRNAs appears to be underestimated and recently reported computational re-analysis of CHO genomic data identified hundreds of additional pre-miR sequences.…”

Sequencing technologies for animal cell culture research

“…The repetitious sequences make de novo assembly of these genomes difficult without a reference genome and only slightly easier when a reference genome is available. A large variety of research pursuits is achievable and within the animal cell technology community, Illumina has been used to sequence the Chinese hamster (Brinkrolf et al 2013;Lewis et al 2013) and CHO Xu et al 2011) genomes, as well as baby hamster kidney and CHO cell line transcriptomes (Johnson et al 2014;Hackl et al 2011). Overall, the Illumina platform is able to produce large amounts of high-quality data via a low-cost, high-throughput methodology.…”