High technical resolution transcriptomics analysis for CHO cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Cricetulus griseus
PROVIDER: GSE100908 | GEO | 2018/02/12
REPOSITORIES: GEO
Similar Datasets
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.
2014-08-04 | GSE57023 | GEO
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: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:Most of the recombinant biotherapeutics employed today to combat severe illnesses e.g. various types of cancer or autoimmune diseases are produced by Chinese hamster ovary (CHO) cells. To meet the growing demand of these pharmaceuticals, CHO cells are under constant development in order to enhance their stability and productivity. The last decades saw a shift from empirical cell line optimization towards rational cell engineering using a growing number of large omics datasets to alter cell physiology on various levels. Especially proteomics workflows reached new levels in proteome coverage and data quality due to advances in high-resolution mass spectrometry instrumentation. One type of workflow concentrates on spatial proteomics by usage of subcellular fractionation of organelles with subsequent shotgun mass spectrometry proteomics and machine learning algorithms to determine the subcellular localization of large portions of the cellular proteome at a given time. Here, we present the first subcellular spatial proteome of a CHO-K1 cell line producing high titers of recombinant antibody and comparison to the spatial proteome of an antibody-producing plasma cell-derived (PCD) myeloma cell line. Both cell lines show strong correlation of immunoglobulin G chains with chaperones and proteins associated in protein glycosylation within the endoplasmic reticulum compartment. However, we report differences in the localization of proteins associated to vesicle-mediated transport, transcription and translation, which may affect antibody production in both cell lines. Furthermore, pairing subcellular localization data with protein expression data revealed elevated protein masses for organelles in the secretory pathway in MPC-11 cells. Our study highlights the potential of subcellular spatial proteomics combined with protein expression as potent workflow to identify characteristics of highly efficient recombinant protein expressing cell lines.
2022-08-02 | PXD029115 | Pride
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 the last years. These studies have aid in the exploration of molecular and cellular mechanisms that could explain cell behavior and to propose new targets to improve production and quality of RP. Some of these transcriptomic and proteomic profiles have been acquired under low temperature, butyrate addition, hyperosomotic pressure, protein degradation phenotypes, cellular engineering, production stability and productive phenotypes. In case of productive phenotypes, several whole cell proteomic studies have been previously reported, where cell populations secreting fusion proteins, monoclonal antibodies (mAb) and antibody fragments, at different specific productivity (Qp), have been compared. However, because of this information has been obtained from whole cell homogenates, highlighted categories represent abundant proteins and have limited coverage of proteome from some organelles such as the classical secretory pathway. Due to the central role of this pathway in protein and lipid metabolism, organelle biogenesis, cell cycle, apoptosis and proliferation, and to be recognized as a bottleneck for protein secretion in mammalian cells, its characterization through subcellular proteomics represents a promising strategy for identification of key targets. In fact, over-expression of some proteins from endoplasmic reticulum and Golgi Apparatus has increased the Qp of HEK293 and CHO cells producing different RP. Recently, proteomic reports of subcellular compartments from a variety of cells have been extensively used to study the protein composition of organelles, protein dynamics and discovering of novel proteins involved in the secretion pathway. Thus, this approach was applied to the secretory pathway of CHO cells to identify novel proteins linked to Qp in the present study. Differential proteomics from all remaining compartments was also carried out, giving new lights over other cellular mechanisms associated to the higher producer phenotype. The novel differentially expressed proteins (DEP) found will improve the engineering strategies of CHO cells that will have a positive impact on titer and quality of RP. Cell lines CHO DP-12 clone #1933 CRL-12444 and clone #1934 CRL-12445, which secrete a mAb against human interleukin 8 (IL-8), were used as cell models of recombinant CHO cell lines producing the same RP at different Qp. Our goal is that the processing of raw proteomics data and the identification and classification of DEP belonging to the classical secretory pathway will allow to propose a model by which the increase in the productivity of CHO cells has been associated with the deregulation of proteins from this pathway that show differential expression among the clones under study. This strategy of subcellular proteomics allowed us to identify a large number of new targets and cell pathways with respect to previous whole cell proteomic studies, which can improve the understanding of the molecular mechanisms behind RP production and provide the basis to design new sub-lines with stronger capabilities.
2021-05-04 | PXD021014 | Pride
Project description:Background: Chinese hamster ovary (CHO) cells are the most widely used mammalian host for recombinant protein production. The primary production method is to construct stable, high-yielding cell lines to provide high-quality, low-cost products. The key to optimizing recombinant protein production is to modify engineered cell lines to obtain better growth, expression, or product quality characteristics. Site-specific integration technology can build monoclonal cell lines with ideal stability, yield, and epigenetics by integrating transgenes at stable transcriptional hotspots, becoming a potential tool for constructing genetically engineered cell lines. The previous study screened the C12orf35 site of CHO cells and built an engineered cell line expressing a mAb by CRISPR/Cas9. While the mechanism of site-specific integration of transgenes to achieve high-efficiency expression also needs to be further studied. In this study, we used the extracellular domains of CLL1 and CD33 as model proteins to construct engineered cell lines integrating at C12orf35. Then we studied the growth, expression, and passage stability of these cell lines and conducted transcriptomic studies. Method: In this study, the transgenes CLL1 and CD33 were site-specifically integrated into the C12orf35 site of the CHO-S cell by CRISPR/Cas9 technology. To obtain stable and high-yielding cell lines, we screened and identified monoclonal cell lines by 5’/3’ junction PCR, out-out PCR, and Western blot. Then we compared the growth characteristics of CLL1 and CD33 monoclonal cell lines with wild-type CHO-S cell lines to investigate the growth characteristics. We studied the protein yield and affinity of CLL1 and CD33 cell lines through protein purification, BCA quantification, and ELISA. In addition, we assessed the stability of monoclonal cell lines in long-term culture by studying the growth characteristics, protein yield, and protein quality of early (5th-10th passage) and late (about 20th-25th passage) cells. We performed transcriptomic studies on cell lines with different transgenes, including CLL1, CD33, and anti-PD-1 mAb, inserted at C12orf35. We analyzed the differential genes, GO enrichment, and KEGG enrichment of cell lines hoping to explore the reasons for the phenotypic changes of cell lines from the transcriptional level, as well as the high expression mechanism of C12orf35 site-specific integration cell lines. Results:Using the site-specific integration technology mediated by CRISPR/Cas9, we successfully built CLL1 and CD33 monoclonal cell lines integrated at the C12orf35 site. We evaluated the cell growth characteristics and protein expression properties. The results showed that the insertion of different transgenes at the C12orf35 site had no specific effect on the growth state of the cells. The proteins yields were about 120 mg/L and 160 mg/L, respectively. And the EC50 values of CLL1 and CD33 with the corresponding antibodies reached about 0.36 μg/mL and 0.1 μg/mL, respectively. In addition, the above indicators had similar properties between the 5th and 20th generations, indicating that the specific integration at C12orf35 can achieve both high expression of the target protein and production stability. Transcriptomic analysis showed that the integration of transgenes at the C12orf35 site of CHO cells mainly induced genes in the GO term of “Regulation of transcription, DNA-templated” differentially expressed. Further analysis screened that ATF4 and multiple ZFPs family members were significantly up-regulated. ATF4 and ZFPs are related to gene transcription and protein synthesis, which may be the mechanism of the stable and high expression of the cell lines. Besides, differential gene analysis showed that Car4 and CXCL12 were significantly up- and down-regulated in CD33 cells, respectively. And KEGG enrichment showed that Wnt, PI3K/AKT, and JAK/STAT pathways related to cell proliferation and apoptosis were significantly enriched in CD33 cells. These genes and pathways may be related to the growth state of CD33. In summary, this paper studied the growth characteristics, protein expression characteristics, and stability of the C12orf35 site-specific integration of recombinant CHO cell lines. Combined with transcriptomic analysis, this paper explored the stable and high expression mechanism of these cell lines, hoping to provide a theoretical reference for the subsequent construction and optimization of high-expression stable cell lines.
2023-05-31 | GSE203219 | 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:Identification of microRNAs specific for high producer CHO cell lines
| PRJNA245229 | ENA