Timecourse RNA Sequencing of three CHO cell lines: non-producing CHOZN®GS-/-, an IgG1 producer, and an EPO-Fc producer
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PROVIDER: PRJNA899862 | ENA |
REPOSITORIES: ENA
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Project description:We analyzed the transcriptomic response in three distinct CHO cell lines: a non-producing host cell line (CHOZN®GS-/-), an immunoglobulin G1 (IgG1) producer, and an erythropoietin Fc fusion (EPO-Fc) producer. We compared the growth and production characteristics of all three cell lines during fed-batch culture. High throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) were used to study differential gene expression analysis of the timecourse dataset with the host cell line CHOZN®GS-/- as the reference. The objective of this analysis was to study the common and unique responses of cell lines producing different protein products. This analysis showed enrichment and transient upregulation of mRNAs involved in ER stress, the UPR, and oxidative protein folding and processing in both protein producers. Analysis of temporal differential expression profiles led to the identification of novel engineering targets.
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Project description:Since few data are available on glycans expressed by human BCRs, we aimed to analyze the Fc glycans of membrane-bound IgG after BCR capture and tryptic digestion by LC-MS. For this purpose, human B-cell lines from Burkitt Lymphoma (Ramos) expressing IgG1-BCRs in the presence or absence of Fc glycans (FcG) were analyzed. The B-cell lines were generated by transduction of the BCR-negative MDL-AID KO cell line with N(297) or mutant Q(297) IgG-BCRs. The BCR sequences used were derived from single-cell sorted human B cells isolated from patients with the autoimmune disease rheumatoid arthritis. Two BCRs were directed towards citrullinated antigens (2G9 and 3F3), while a third BCR was directed against tetanus toxoid (D2). In addition, we analyzed the Fc glycan profile of IgG secreted (sIgG) by human Ramos B cells for comparison.
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Project description:CD34+ progenitors were isolated from the bone marrow of three healthy volunteers. CD34+CD71+CD45RA- were FACS sorted to enrich for erythroid progenitors. The cells were cultured for four hours with or without EPO in combination with LY294002, and harvested for RNA extraction, amplification and expression analysis. A compound treatment design type is where the response to administration of a compound or chemical (including biological compounds such as hormones) is assayed. Compound Based Treatment: EPO Keywords: compound_treatment_design
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Project description:Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.
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Project description:Erythropoietin (EPO) drives erythropoiesis and is secreted mainly by the kidney upon hypoxic or anemic stress. The paucity of EPO production in renal EPO-producing cells (REPs) causes renal anemia, one of the most common complications of chronic nephropathies. Although mitochondrial dysfunction is commonly observed in several renal and hematopoietic disorders, the mechanism by which mitochondrial quality control impacts renal anemia remains elusive. In this study, we showed that FUNDC1, a mitophagy receptor, plays a critical role in EPO-driven erythropoiesis induced by stresses. Mechanistically, EPO production is impaired in REPs in Fundc1-/- mice upon stresses, and the impairment is caused by the accumulation of damaged mitochondria, which consequently leads to the elevation of the reactive oxygen species (ROS) level and triggers inflammatory responses by up-regulating proinflammatory cytokines. These inflammatory factors promote the myofibroblastic transformation of REPs, resulting in the reduction of EPO production. We therefore provide a link between aberrant mitophagy and deficient EPO generation in renal anemia. Our results also suggest that the mitochondrial quality control safeguards REPs under stresses, which may serve as a potential therapeutic strategy for the treatment of renal anemia.
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Project description:The mechanisms involved in the maintenance of memory IgE responses are poorly understood, and the role played by germinal center (GC) IgE cells in these memory responses is particularly unclear. IgE B-cell differentiation is characterized by a transient GC phase, a bias towards the plasma cell (PC) fate, and dependence on sequential switching for the production of high-affinity IgE. We show here that IgE GC B cells are unfit to undergo the conventional GC differentiation program due to impaired B-cell receptor function and increased apoptosis. IgE GC cells fail to populate the GC light zone and are unable to contribute to the memory and long-lived PC compartments. Furthermore, we demonstrate that direct and sequential switching are linked to distinct B-cell differentiation fates: direct switching generates IgE GC cells, whereas sequential switching gives rise to IgE plasma cells. We propose a comprehensive model for the generation and memory of IgE responses. The purpose of this analysis was to: 1) identify expression differences between IgE and IgG1 B lymphocytes, 2) identify GC Dark Zone (DZ) and Light Zone (LZ) signatures of IgG1 GC cells. For that purpose, we compared in one experiment the gene expression patterns of IgE germinal center (GC) cells, IgG1 GC cells, IgE plasma cells (PC), IgG1 PC and naïve cells. In a second experiment, we compared the expression of IgG1 DZ GC cells with that of IgG1 LZ GC cells. Triplicates obtained from independent sorting experiments were used for all samples except two (IgG1 PC=2 samples; IgE PC=4 samples). Each sample was obtained from a pool of three individual mice. The mice used in the experiment were CeGFP BALB/c mice infected with the parasite N. brasiliensis. CeGFP mice carry an IRES-GFP KI cassette in the 3'UTR of membrane IgE. In these mice, GFP expression marks IgE cells, and a population of IgG1 cells with a rearrangement to Cepsilon in the non-productive (VDJ negative) IgH chromosome.
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