Identification of microRNAs specific for high producer CHO cell lines
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Rattus norvegicus Homo sapiens Cricetulus griseus
PROVIDER: GSE57023 | GEO | 2014/08/04
SECONDARY ACCESSION(S): PRJNA245229
REPOSITORIES: GEO
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Project description:Mature microRNA levels were analyzed in 4 producing CHO cell lines expressing 2 structurally different recombinant proteins at low and high level as well as in 1 non-producing CHO cell line. The goal was to identify microRNAs that are involved in heterologous protein formation and secretion. 5 recombinant CHO cell lines. 3 biological replicates each. All samples were hybridized against a common reference (pool of all total RNA samples).
2014-08-04 | E-GEOD-57023 | biostudies-arrayexpress
Project description:We present the first comprehensive genome-wide view of CHO translational activity during recombinant expression by the application of ribosome profiling. The distribution of translational power in CHO cells was analyzed in the context of the recombinant mRNAs and the endogenous mRNA pool. In an antibody-producing CHO cell line, the recombinant mRNAs were found to be the most abundant transcripts and also sequestered a substantial amount of translating ribosomes (up to 15%). The recombinant mRNAs were translated as efficiently as the endogenous mRNAs, and changes in translation and transcription of the recombinant mRNAs were directly reflected in changes in specific productivity. Interestingly, improvements in bioprocess quality attributes were achieved by depleting the highly expressed and translated inert NeoR mRNA by siRNA-mediated knock-down. This resulted in improved cellular growth, which was accompanied by an 18% increase in antibody titers. This study is the first to carefully map the translatome of the CHO cell to the nucleotide level and to demonstrate how recombinant expression affects the cell on the translational level.
2016-12-21 | GSE79512 | GEO
Project description:Biologics have emerged as leading blockbuster therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here we identify bottlenecks limiting expression of challenging recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293. Surprisingly, one third of the challenging human proteins displayed an improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, a key set of genes with extreme expression variation was identified. Among these, ATF4 and SRP9 were validated as general productivity boosters in CHO. Further, we found that more heavily glycosylated products benefit more from the elevated activities of the N- and O-glycosyltransferases found in HEK293 cells. Collectively, our results demonstrate the utilization of HEK293 cells for expression rescue of human proteins and suggest a strategy for identification of secretory pathway components improving yield and quality in HEK293 and CHO.
2022-06-22 | GSE157729 | GEO
Project description:We developed an artificial gene expression system with transcriptional positive-feedback loop of transactivator to induce the high expression of target genes in CHO cells. DNA microarray analysis was conducted in semi-continuous culture with high-cell-density culture under normal and low temperature conditions of recombinant scFv-Fc-producer CHO cells (CHO/aTF_scFv-Fc2 cells).
2024-01-01 | E-MTAB-13440 | biostudies-arrayexpress
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. 36 miRNA libraries from three different CHO cell lines and two process condition. In the control run temperature was maintained at 30°C, while temperature was reduced to 30°C after reaching mid exponential phase
2016-04-03 | E-GEOD-72945 | biostudies-arrayexpress
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
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
Project description:Mature microRNA transcripts were analyzed in 9 CHO cell lines growing at different proliferation rates. The goal was to identify microRNAs that correlate positively or negatively with CHO proliferation rates.
2015-12-10 | GSE75830 | GEO
Project description:mature microRNA transcripts were analyzed in 5 CHO cell lines growing at different proliferation rates. The goal was to identify microRNAs that correlate positively or negatively with CHO proliferation rates.
2014-01-07 | GSE52994 | GEO
Project description:Filamentous fungi are important factories in the production and secretion of homologous and heterologous lignocellulolytic enzymes, however the regulation of protein secretion in these organisms need to be further explored. In order to investigate this regulation, Aspergillus nidulans recombinant strains were analyzed by transcriptomics. We designed three A. nidulans recombinant strains producing the following heterologous proteins: alpha-arabinofuranosidase (AbfA), beta-glucosidase (BglC) and thermophilic mannanase (1542). The heterologous genes abfA and bglC were highly expressed, while the levels of 1542 mRNA were similar to the reference gene. An indirect relationship between mRNA and the levels of protein secretion was observed, suggesting that transcription is not a bottleneck in these systems. Based on the general analysis of RNA-seq of the recombinant strains, it was possible to observe a predominant up-regulation response. Moreover, biological processes related to metabolism, protein with binding function and cellular transport were overrepresented. We also observed the unconventional splicing of hacA for the recombinant A. nidulansAbfA and A. nidulans1542, indicating some level of unfolded protein response. The global analysis showed mild stress at 2 h induction of heterologous protein production, which was normalized after 8 h. Our results provide insights to understand how A. nidulans adapts to the overproduction of heterologous proteins.
2020-07-14 | GSE101522 | GEO