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CHO smallRNA sequencing for microRNA annotation

ABSTRACT: MicroRNAs are small non-coding RNAs that play a critical role in post-transcriptional control of gene expression. Recent publications of genomic sequencing data from CHO cells provide new tools for the discovery of novel miRNAs in this important production system. The current release of the miRNA registry miRBase contains 307 mature miRNAs and 200 precursor sequences. We searched for evolutionary conserved miRNAs in recently published genomic data, derived from hamster and CHO cells, to further extend the list of known miRNAs. With our approach we could identify several dozens novel miRNAs and verify their expression with multiple next-generation sequencing experiments. In addition, a great amount of unexpressed miRNAs could be found, awaiting further confirmation by testing for their transcription in different Chinese hamster tissues.

ORGANISM(S): Cricetulus griseus

PROVIDER: GSE59838 | GEO | 2015/03/02

SECONDARY ACCESSION(S): PRJNA256330

REPOSITORIES: GEO

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CHO smallRNA sequencing for microRNA annotation

Project description:MicroRNAs are small non-coding RNAs that play a critical role in post-transcriptional control of gene expression. Recent publications of genomic sequencing data from CHO cells provide new tools for the discovery of novel miRNAs in this important production system. The current release of the miRNA registry miRBase contains 307 mature miRNAs and 200 precursor sequences. We searched for evolutionary conserved miRNAs in recently published genomic data, derived from hamster and CHO cells, to further extend the list of known miRNAs. With our approach we could identify several dozens novel miRNAs and verify their expression with multiple next-generation sequencing experiments. In addition, a great amount of unexpressed miRNAs could be found, awaiting further confirmation by testing for their transcription in different Chinese hamster tissues. Two batch processes with 6 samples each

2015-03-02 | E-GEOD-59838 | biostudies-arrayexpress

A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells

Project description:Gene expression is a key determinant of phenotypes that made Chinese Hamster Ovary (CHO) cells, with their human-like glycosylation profile and high protein titers, one of the most widely used cells for the production of therapeutic proteins and biopharmaceuticals. Engineering CHO gene expression thus holds a key to improve drug quality and cost effective production. However, the success of engineering gene expression or ectopic activation of silent genes to optimize desired pathways requires accurate annotation of the underlying regulatory elements and the transcription start site (TSS). Unfortunately, to date, most TSSs of CHO-expressed genes and the ~50% of hamster genes that are silent in CHO were computationally predicted and are frequently inaccurate. To oust this hurdle, we report revised TSSs annotations for 15,308 Chinese Hamster genes and 4,145 non-coding RNAs based on experimental data from CHO K1 cells and 10 hamster tissues. In the example of the glycosyltransferase gene Mgat3, we further demonstrate how accurate annotations readily facilitate activating silent genes by CRISPRa. Together, we envision that our annotation and data from the Chinese Hamster will provide a rich resource for the CHO community, improve genome engineering efforts and additionally aid comparative and evolutionary studies.

2020-10-06 | GSE159044 | GEO

Comparative genomic hybridization of CHO Chr6 cell line

Project description:Aim is to validate content of human genomic material in CHO cells monosomal for human chromosome 6. Comparative genomic hybridization to define human genomic regions maintained in hamster hybridoma cell line.

2014-06-05 | E-GEOD-56334 | biostudies-arrayexpress

Comparison of transcriptional profile of CHO cells cultured in different media.

Project description:Two clones of Chinese hamster ovary (CHO) cells were cultured in 2 different chemical difined custom media. Transcriptional profile were compeared between clones and media.

2022-03-01 | GSE197570 | GEO

Profiling miRNAs of high, low and non-producing CHO cells during biphasic Fed-Batch cultivations reveal interesting targets for cell line engineering

Project description:Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production mode for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where culture temperature is decreased to maximize volumetric product yields. However, it still remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes since single miRNAs are capable of regulating entire physiological pathways. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37 versus 30 Â°C culture temperature. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which might be interesting for CHO cell engineering. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes such as recombinant protein production, apoptosis, necrosis and proliferation. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. Taken together, our study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch process and functionally evaluated their potential for host cell engineering. 36 miRNA libraries from three different CHO cell lines and two process condition. In the control run temperature was maintained at 30Â°C, while temperature was reduced to 30Â°C after reaching mid exponential phase

2016-04-03 | E-GEOD-72945 | biostudies-arrayexpress

Profiling miRNAs of high, low and non-producing CHO cells during biphasic Fed-Batch cultivations reveal interesting targets for cell line engineering

Project description:Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production mode for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where culture temperature is decreased to maximize volumetric product yields. However, it still remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes since single miRNAs are capable of regulating entire physiological pathways. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37 versus 30 °C culture temperature. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which might be interesting for CHO cell engineering. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes such as recombinant protein production, apoptosis, necrosis and proliferation. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. Taken together, our study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch process and functionally evaluated their potential for host cell engineering.

2016-04-03 | GSE72945 | GEO

High technical resolution transcriptomics analysis for CHO cells

Project description:The development of next-generation sequencing technologies has opened new opportunities to better characterize complex eukaryotic cells. Chinese hamster ovary (CHO) cells play a primary role in therapeutic protein production, with currently five of the top ten blockbuster selling drugs produced in CHO. However, engineering superior CHO cells with improved production features has had limited results to date and cell lines are still developed through generation and screening of large strain pools. Here, we applied RNA sequencing to contrast a high and a low monoclonal antibody producing cell line. Rigorous experimental design achieved high reproducibility between biological replicates, remarkably reducing variation to less than 10%. More than 14,000 gene-transcripts were identified and surprisingly 58% were classified as differentially expressed, including 2,900 with a fold change higher than 1.5. The largest differences were found for gene-transcripts belonging to regulation of apoptosis, cell death and protein intracellular transport GO term classifications, which were found to be significantly enriched in the high producing cell line. RNA sequencing was also performed to lower producing subclones, where down-regulation of genes encoding secreted glycoproteins was found to be the most significant change. The large number of significant differences even between subclones suggests a substantial genetic fluidity in CHO lines and challenges the notion of identifying and manipulating a few key genes to generate high production CHO cell lines.

2018-02-12 | GSE100908 | GEO

Defining lncRNAs Associated with CHO Cell Growth and IgG Productivity by RNA-Seq II

Project description:Small-scale bioreactors used in Chinese hamster ovary (CHO) cell line development allow transcriptomic studies on multiple cell lines. Here we define the CHO cell long non-coding RNA (lncRNA) transcriptome from cells grown in controlled miniature bioreactors under fed-batch conditions using RNA-Seq to identify lncRNAs and how the expression of these changes throughout growth and between IgG producers. We identify lncRNAs associated with productivity and growth characteristics, finding that Adapt15, linked to ER stress, GAS5, linked to mTOR signalling/growth arrest, and PVT1, linked to Myc expression, are differentially regulated during fed-batch culture and whose expression relates to productivity and growth. Changes in (non)-coding RNA expression between the seed train and the equivalent day of fed-batch culture are also reported, showing large differences in gene expression between these, and compared with existing datasets. Collectively, we present a comprehensive lncRNA CHO cell profiling and identify targets for engineering growth and productivity characteristics of CHO cells.

2019-11-20 | GSE140669 | GEO

Defining lncRNAs Associated with CHO Cell Growth and IgG Productivity by RNA-Seq I

2019-11-20 | GSE140668 | GEO

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Project description:The effects of changes in bioprocessing parameters (temp, pH, DO) on anti-IL8 IgG1 producing Chinese hamster ovary (CHO) cells were studied on the level of host cell proteome expression using TMT-SPS-MS3.

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