Revealing suspension adapation of CHO cell in serum free media
Ontology highlight
ABSTRACT:
ORGANISM(S): Cricetulus griseus
SUBMITTER: Namil Lee
PROVIDER: E-GEOD-75094 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Purpose : We performed strand-specific RNA-seq analysis of CHO cells according to adaptation trajectory to identify the specific genes responded to the suspension adaptation. Result : Cell growth, replication, and nucleotide metabolism categories were enriched in up regulated clusters whereas cellular community category which contains focal adhesion related genes were enriched in down regulated clusters
2015-11-19 | GSE75094 | GEO
Project description:A comprehensive Chinese hamster ovary (CHO)-cell specific spectral library, containing extensive information of high-quality spectral ions covering more than 10k CHO cell proteins, was constructed to provide confident and reproducible protein identification and quantification in data-independent SWATH-MS analysis. The applicability of CHO spectral library was tested in the analyses of different CHO samples including whole cell lysate, harvested cell culture fluids, and downstream processing samples. The portability of CHO spectral library was also demonstrated in the processing and analyses of SWATH-MS data sets collected from multiple LC-MS instrumental setups and various CHO cell lines.
2020-07-20 | PXD016047 | Pride
Project description:γ-secretase is an intra-membrane-cleaving aspartyl protease implicated in the processing of a wide range of type I membrane proteins including the Notch receptor and the amyloid-β precursor protein (APP). It thus regulates a diverse array of cellular and biological processes including the differentiation of neuronal embryonic stem cells, or intestinal stem cells, with the latter controlling the self-renewing of the intestinal epithelium. Indeed, proteolysis of these proteins by γ-secretase triggers signaling cascades by releasing intracellular domains (ICDs) which, following association with adaptor proteins and nuclear translocation, modulate the transcription of different genes by binding directly to their promoters. The pronounced proliferative and regenerative effects of Notch signaling and its implication in the generation of the Aβ-peptides, makes γ-secretase a therapeutic target for several types of cancer and for Alzheimerâs disease. To investigate the broad effects of γ-secretase activity onto the cellular transcriptome, Chinese hamster ovary (CHO) cells with enhanced γ-secretase were compared to cells with abolished γ-secretase activity via a microarray designed for a genetically close species, mouse. Our findings will potentially help to decipher the biology of γ-secretase, including a better understanding of the roles of this enzyme in gene transcription. We compared the transcriptomes of two CHO cell lines displaying extreme differences in γ-secretase activity. The S-1 cell line overexpressed the four components of γ-secretase (NCT, APH1aL, PS1, and PEN2) and was characterized by a marked increase in the level of PS1 heterodimers associated with 8-fold increased γ-secretase activity compared to untransfected controls. The other cell line consisted of wild type CHO cells incubated with DAPT, a well-known γ-secretase inhibitor. The two cell lines were used in combination with a mouse microarray to analyze gene transcription under enhanced γ-secretase. Two samples, S1 and DAPT treated CHO- were used in biological triplicates each.
2010-05-15 | E-GEOD-16379 | biostudies-arrayexpress
Project description:Transcriptional profiling of CHO-K1 cells comparing to in-house serum-free and suspension adapted CHO-K1 cells in the exponential phase. Goal was to determine the effects of serum on CHO-K1 cells.
2016-12-24 | GSE92885 | GEO
Project description:Chinese hamster ovary (CHO) cells have been widely employed for production of recombinant proteins (RP) for research and therapeutic purposes. Indeed, this expression system was used for production of most of approved antibodies (84%) in 2015-2018 period. The success of this cell line for RP production relies on several advantages that comprise but are not limited to a safety viral profile, human compatible glycosylation, development of specific culture mediums and supplements, availability of sub-lines with different capabilities and the improvement in design of DNA vectors and selection strategies that allows to obtain higher producer clones in an easier way. Given the high importance of this cell line, a deeper knowledge of their biology through genomic, transcriptomic, proteomic and metabolomic studies has been gained in last years. These studies have aid in the exploration of molecular and cellular mechanisms that could explain cell behavior and to propose new targets for improved RP production and quality. A special emphasis has been placed on proteomic characterization of subcellular compartments over cellular homogenates, due to some advantages that fractionation techniques offer over the classical approach, which include obtaining datasets that are easier to process and understand. Subcellular proteomics allows monitoring of proteins that shuttle between different compartments, quantification of lower abundance proteins, increase in number of identified proteins, elucidation of function and regulation of proteins, and unraveling of organelle dynamics. The use of subcellular fractionation for proteomic characterization of organelles from CHO cells has been limited to the isolation of one or few organelles in an adherent phenotype, and only few fractionation protocols have been reported for these cells in their suspension format. The high cross-contamination of some isolated fractions due to insufficient fractionation steps, prevents adopting some of these protocols for applications such as proteomics. Thus, since this approach has not been addressed before for the proteomic characterization of CHO cell organelles, subcellular proteomics was harnessed in the present study to provide identification and quantification of proteins from subcellular compartments obtained by differential and isopycnic centrifugation of this cell line. Although several cellular processes and organelles play an important role during expression and secretion of RP, the classical secretion pathway has been recognized as a bottleneck for post-translational modifications, transport, modification and secretion of proteins in mammalian cells. In fact, over-expression of some proteins from endoplasmic reticulum and Golgi Apparatus has increased the specific productivity of HEK293 and CHO cells producing different RP. Thus, in line with the paramount importance of organelles from classical secretion pathway for RP production, a novel discontinuous sucrose gradient for improved separation and enrichment of microsomes has been designed in our laboratory, and incorporated to the proteomic characterization of CHO cell organelles. CRL-12444, a DP-12 derived cell line producing a humanized monoclonal antibody against human IL-8, was used as a model of a recombinant CHO cell line during this study. Our goal is that the processing of raw proteomics data, the classification and mapping of identified proteins will allow to construct proteomic maps for most subcellular compartments, describing the molecular functions, biological processes and pathways from enriched fractions. The identification of proteins in each enriched compartment will improve the knowledge about their protein components, function and interaction, especially from those poorly represented like the proteins in the microsomes, allowing a subsequently expansion of the engineering strategies of CHO cells to increase the titer and quality of RPs.
2020-08-27 | PXD019778 | Pride
Project description:Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is the workhorse of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics properties is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell clones and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.
2021-05-15 | E-MTAB-10417 | biostudies-arrayexpress
Project description:We have repurposed standard phosphoproteomics workflow based on Fe3+IMAC for enrichment ofmannose-6-phosphate modified glycopeptides. This worklfow was used to profile lysosomal acid hydrolases in HeLa and CHO cell lines. We have combined this approach with CRISPR/Cas9 KO of acid phosphatases 2 and 5 responsible for dephosphorylation of mannose-6-phosphate glycopeptides in the lysosome. This combined approach enabled significantly deeper coverage of CHO mannose-6-phosphate glycoproteome.
2018-12-18 | PXD010333 | Pride
Project description:The yeast Saccharomyces cerevisiae is a highly powerful model for systems genetics. While the advent of ordered deletion libraries has considerably facilitated yeast screening, it has also narrowed the diversity of screenable variants and precluded the exploration of the non-coding genome. Here, we present a versatile, time- and work-efficient method to functionally explore the yeast genome at an unprecedented throughput and resolution, using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows the one-step mapping of all genetic loci in which a transposon can be inserted without disrupting a function necessary for cell growth. SATAY is especially suited to discover loci important for growth in various conditions. Here, we demonstrate that SATAY can be used to (2) reveal genetic interactions in single and multiple mutant strains, (1) reveal drug-resistant and -sensitive mutants, (3) detect not only essential genes, but also essential functional protein domains, and (4) generate not only null alleles, but also other informative genetic variants. Thus SATAY allows to easily explore the yeast genome at an unprecedented resolution and throughput.
2017-05-11 | E-MTAB-4885 | biostudies-arrayexpress
Project description:Revealing suspension adapation of CHO cell in serum free media
| PRJNA302601 | ENA
Project description:Polyamines, including putrescine (PUT), spermidine (SPD) and spermine (SPM), are abundant polycations supporting various cellular functions. Their cellular content is tightly controlled by biosynthesis, degradation and uptake or export via the polyamine transport system (PTS). However, the mammalian PTS remains poorly characterized. Mutated Chinese hamster ovary cells, CHO-MG, are frequently used to study the PTS owing to their phenotype of reduced polyamine uptake and resistance to methylglyoxal bis-(guanylhydrazone) (MGBG), a toxic polyamine biosynthesis inhibitor. Yet, the underlying genetic defect remains enigmatic. We recently proved that the P5B-type ATPase, ATP13A2, is a polyamine transporter and here, we analyzed whether the P5B-type ATPases are deficient in CHO-MG. Label-free shogun proteomics found that ATP13A3 expression is reduced in CHO-MG. Mutations in the ATP13A3 gene were found to critically disturb the protein sequence. Interestingly, depleting ATP13A3 in the wild-type CHO cells induced a CHO-MG phenotype, whereas its complementation in CHO-MG rescued the phenotype. Together, we demonstrate that defective ATP13A3 contributes to the CHO-MG phenotype, designating ATP13A3 as a new member of the mammalian PTS that may partially overlap in function with ATP13A2.
2020-12-09 | PXD020559 | Pride