Nanopore Cas9-targeted sequencing in CHO
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PROVIDER: PRJEB57448 | ENA |
REPOSITORIES: ENA
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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
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
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.
2021-04-09 | PXD017779 | Pride
Project description:In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the most commonly used host cell for recombinant protein production, especially antibodies. Over the last decade in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be an ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we sequenced non-coding RNA of Exosomes (EXO) and whole cell lysate (WCL) isolated from CHO-K1 Cell Cultures at different growth phases (logarithmic/exponential phase (log/exp), stationary phase (stat), as well as death phase at 80 % viability (80 % ) and 60 % viability (60 %)) via Lexogen Small RNA-Seq Library Prep Kit for Illumina on the Illumina MiSeq platform in PE mode 2 x 36nt.
2021-01-18 | E-MTAB-9894 | biostudies-arrayexpress
Project description:Profiling of conserved microRNAs in Chinese hamster ovary (CHO) cell lines
| PRJNA80861 | ENA
Project description:The majority of recombinant protein therapeutics are produced with Chinese Hamster Ovary (CHO) cells. Productivity depends on the initial cell line engineering in terms of integration site or choice of an appropriate promotor for the recombinant gene expression, as well as media and process parameter optimization. Here, proteomic profiling is used to identify optimization targets for a pharmaceutical relevant cell line system. Triplicates of CHO-K1 cell 2 L bioreactor fedbatch cultivations were performed and daily sampled for nLC-MS/MS proteomic analysis. Collected data from day 3 up to day 11 showed high Pearson correlation of 93.7 ± 4 % with ca. 2500 proteins quantified. The different growth phases were separated by principal component analysis and hierarchical clustering approaches. Subsequent statistical analysis revealed distinct protein profiles, where steady increase or decrease over time were the most prominent clusters. Fisher exact enrichment tests yielded in significantly enriched protein annotations which were successfully mapped to growth and metabolic changes during fedbatch cell cultivation. Major improvements in cellular and process understanding were achieved and yielded in the identification of promising new targets, like strong endogenous promotors, for cellular engineering and process optimization of this biopharmaceutical relevant cell line.
2021-01-25 | PXD018439 | Pride
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: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:Chinese Hamster ovary (CHO) cells are the main platform used to produce recombinant proteins in the biopharmaceutical industry and are commonly cultured in either fed-batch or perfusion mode. However, the optimization of the complex biological systems used in such processes is extremely challenging. Omics approaches can reveal otherwise unknown characteristics of these systems and identify culture parameters that can be manipulated to optimize the cultivation process. Here we have applied proteomic profiling to a monoclonal antibody (mAb) production operated in perfusion mode to explore how cell biology and reactor environment change as the cell density reaches ≥ 200 x 106 cells/mL. The proteomics data show an increase of structural proteins as cell density increase, signs of oxidative stress and changes in glutathione metabolism at very high cell densities. Additionally, metabolomic profiling was carried out. See article “High cell density culture has a maintained exoproteome and metabolome” for more information.
2018-07-04 | PXD008760 | Pride
Project description:Thermal hysteresis in CHO K1, a Chinese Hamster (Cricetulus griseus) Ovary derived established cell line
| PRJDB7873 | ENA