Plasma Membrane Effects of Sphingolipid-Synthesis Inhibition by Myriocin in CHO Cells: A Biophysical and Lipidomic Study

Abstract Two main strategies for establishing the cellular effects of a given enzyme activity suppression are (a) the use of a stably mutated cell line that lacks a functional gene, or (b) treating the wild type with an inhibitory compound that affects the same gene-product protein. In this work, myriocin was used to block the serine palmitoyltransferase (SPT) enzyme of CHO cells and the subsequent biophysical changes in membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) happened to be lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated glycerophospholipids than the non-treated ones, although they achieved it only partially, their plasma membranes remaining more fluid and less penetrable than those from the control cells.

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Development of a Non-protein and Lipid Medium Adopted Cell Line for Biopharmaceutical Recombinant Protein Expression

The Open Biotechnology Journal ◽

10.2174/1874070701307010001 ◽

2013 ◽

Vol 7 (1) ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Tetsuji Sasaki ◽

Akiyoshi Taniguchi

Keyword(s):

Growth Factors ◽

Recombinant Protein ◽

Cell Line ◽

Cho Cells ◽

Recombinant Protein Expression ◽

Culture Method ◽

Luciferase Reporter ◽

Cho Cell ◽

Reporter System ◽

The Cost

Recently, many biopharmaceuticals have been developed such as cytokines, growth factors, and antibodies. These recombinant proteins are mostly expressed by CHO cells. However, the culture medium of CHO cells requires the addition of serum, which can contain unknown biological substances such as viruses, or requires the addition of expensive growth factors. To avoid the risks of biological ingredients and to decrease the cost of biopharmaceutical production, we developed a non-protein and lipid medium adopted (NPLAd) CHO cell line using the adapted culture method. Our results indicated that autocrine EGF production and insulin addition are essential for NPLAd CHO cell growth. However, the rate of cell proliferation of NPLAd CHO cells was decreased compared with original CHO-K1 cells. The proliferation of NPLAd CHO cells was improved by GM3 addition, suggesting increased signaling efficiency of autocrine factors. No difference was found in the growth rate between original CHO-K1 and NPLAd CHO cells supplemented with insulin and GM3. The productivity of recombinant protein in NPLAd CHO cells was verified using secreting luciferase reporter system. As a result, luciferase activity in NPLAd CHO cells showed more than three times higher than in the original CHOK1 cells. The results suggested that this cell line could be useful for biopharmaceutical recombinant protein.

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Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22052407 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2407

Author(s):

Sung Wook Shin ◽

Dongwoo Kim ◽

Jae Seong Lee

Keyword(s):

Cell Line ◽

Chinese Hamster Ovary ◽

Cho Cells ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Cho Cell ◽

Cell Line Development ◽

Cho Cell Line ◽

Vector Systems ◽

Targeted Integration

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.

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Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine for production

10.1101/317537 ◽

2018 ◽

Author(s):

Sara M O’Rourke ◽

Gabriel Byrne ◽

Gwen Tatsuno ◽

Meredith Wright ◽

Bin Yu ◽

...

Keyword(s):

Sialic Acid ◽

Cell Line ◽

Cell Lines ◽

Cho Cells ◽

Vaccine Development ◽

Rapid Development ◽

Hiv Vaccine ◽

Antigenic Structure ◽

Cho Cell ◽

Cho Cell Line

AbstractThe production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10-100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel gene-edited CHO cell line (MGAT1-CHO) to address the problems of low expression, high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1-CHO cell line. We also show that the efficiency of this process can be greatly improved with robotic selection. Finally, we describe a MGAT1-CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years.

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Receptor-mediated stimulation of lipid signalling pathways in CHO cells elicits the rapid transient induction of the PDE1B isoform of Ca2+/calmodulin-stimulated cAMP phosphodiesterase

Biochemical Journal ◽

10.1042/bj3210157 ◽

1997 ◽

Vol 321 (1) ◽

pp. 157-163 ◽

Cited By ~ 12

Author(s):

Sandra SPENCE ◽

Graham RENA ◽

Michael SULLIVAN ◽

Suat ERDOGAN ◽

Miles D. HOUSLAY

Keyword(s):

T Cells ◽

Reverse Transcriptase ◽

Cell Line ◽

Cho Cells ◽

Cho Cell ◽

Degenerate Primers ◽

Jurkat T Cells ◽

Camp Phosphodiesterase ◽

Reverse Transcriptase Pcr ◽

Cho Cell Line

Chinese hamster ovary cells (CHO cells) do not exhibit any Ca2+/calmodulin-stimulated cAMP phosphodiesterase (PDE1) activity. Challenge of CHO cells with agonists for endogenous P2-purinoceptors, lysophosphatidic acid receptors and thrombin receptors caused a similar rapid transient induction of PDE1 activity in each instance. This was also evident on noradrenaline challenge of a cloned CHO cell line transfected so as to overexpress α1B-adrenoceptors. This novel PDE1 activity appeared within about 15 min of exposure to ligands, rose to a maximum value within 30 min to 1 h and then rapidly decreased. In each case, the expression of novel PDE1 activity was blocked by the transcriptional inhibitor actinomycin D. Challenge with insulin of either native CHO cells or a CHO cell line transfected so as to overexpress the human insulin receptor failed to induce PDE1 activity. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1C isoform, did not amplify any fragment from RNA preparations of CHO cells expressing PDE1 activity, although they did so from the human thyroid carcinoma FTC133 cell line. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1A and PDE1B isoforms, successfully amplified a fragment of the predicted size from RNA preparations of both CHO cells expressing PDE1 activity and human Jurkat T-cells. Sequencing of the PCR products, generated using the PDE1A/B primers, yielded a novel sequence which, by analogy with sequences reported for bovine and murine PDE1B forms, suggests that the PDE1 species induced in CHO cells through protein kinase C activation and that expressed in Jurkat T-cells are PDE1B forms.

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Study on the mammalian cell lines of stable expression strain matrix protein of rabies virus CTN-1V

Journal of Applied Virology ◽

10.21092/jav.v5i4.82 ◽

2017 ◽

Vol 5 (4) ◽

pp. 65

Author(s):

Yunpeng Wang ◽

Shouchun Cao ◽

Jia Li ◽

Jianrong Tang ◽

Leitai Shi ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Rabies Virus ◽

Cho Cells ◽

Matrix Protein ◽

Recombinant Expression ◽

Cho Cell ◽

Mammalian Cell Lines ◽

Cho Cell Line ◽

The Matrix

Objective: To construct CHO cell line stably expressing rabies virus matrix protein. Methods: RT-PCR was used to amplify and isolate CTV-1V gene of rabies virus. After cloning into pCDNA5.0FRT vector, the recombinant plasmid pCDNA5.0FRT-M was constructed and then transfected into CHO cells with POG44 plasmid. The positive clones were screened by hygromycin B and the stable cell lines were identified by indirect immunofluorescence and Western blot. Results:After enzyme digestion and DNA sequencing, the recombinant expression plasmid pCDNA5.0FRT-M were transfected into CHO cells, get the visible positive cell clones, scraping positive clones were expanded in culture and defined as the second generation, After 10 generations, the results were still positive.Conclusion The CHO cell line stably expressing rabies virus matrix protein was successfully established, which lays a foundation for further study of the function of the matrix protein.

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Using Metabolomics to Identify Cell Line-Independent Indicators of Growth Inhibition for Chinese Hamster Ovary Cell-Based Bioprocesses

Metabolites ◽

10.3390/metabo10050199 ◽

2020 ◽

Vol 10 (5) ◽

pp. 199 ◽

Cited By ~ 2

Author(s):

Nicholas Alden ◽

Ravali Raju ◽

Kyle McElearney ◽

James Lambropoulos ◽

Rashmi Kshirsagar ◽

...

Keyword(s):

Growth Inhibition ◽

Cell Line ◽

Cell Lines ◽

Chinese Hamster Ovary ◽

Cho Cells ◽

Feeding Strategy ◽

Chinese Hamster ◽

Ovary Cell ◽

Growth Stages ◽

Cho Cell

Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals. Efforts to improve productivity through medium design and feeding strategy optimization have focused on preventing the depletion of essential nutrients and managing the accumulation of lactate and ammonia. In addition to ammonia and lactate, many other metabolites accumulate in CHO cell cultures, although their effects remain largely unknown. Elucidating these effects has the potential to further improve the productivity of CHO cell-based bioprocesses. This study used untargeted metabolomics to identify metabolites that accumulate in fed-batch cultures of monoclonal antibody (mAb) producing CHO cells. The metabolomics experiments profiled six cell lines that are derived from two different hosts, produce different mAbs, and exhibit different growth profiles. Comparing the cell lines’ metabolite profiles at different growth stages, we found a strong negative correlation between peak viable cell density (VCD) and a tryptophan metabolite, putatively identified as 5-hydroxyindoleacetaldehyde (5-HIAAld). Amino acid supplementation experiments showed strong growth inhibition of all cell lines by excess tryptophan, which correlated with the accumulation of 5-HIAAld in the culture medium. Prospectively, the approach presented in this study could be used to identify cell line- and host-independent metabolite markers for clone selection and bioprocess development.

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Glycoproteomic Characterization of FUT8 Knock-Out CHO Cells Reveals Roles of FUT8 in the Glycosylation

Frontiers in Chemistry ◽

10.3389/fchem.2021.755238 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ganglong Yang ◽

Qiong Wang ◽

Lijun Chen ◽

Michael J. Betenbaugh ◽

Hui Zhang

Keyword(s):

Cell Line ◽

Relative Abundance ◽

Cho Cells ◽

Gene Knockout ◽

Protein Glycosylation ◽

Wild Type ◽

Cho Cell ◽

Knock Out ◽

Cho Cell Line

The α1,6-fucosyltransferase (encoded by FUT8 gene) is the key enzyme transferring fucose to the innermost GlcNAc residue on an N-glycan through an α-1,6 linkage in the mammalian cells. The presence of core fucose on antibody Fc region can inhibit antibody-dependent cellular cytotoxicity (ADCC) and reduce antibody therapeutic efficiency in vivo. Chinese hamster ovary (CHO) cells are the predominant production platform in biopharmaceutical manufacturing. Therefore, the generation of FUT8 knock-out (FUT8KO) CHO cell line is favorable and can be applied to produce completely non-fucosylated antibodies. The characterization of monoclonal antibodies as well as host cell glycoprotein impurities are required for quality control purposes under regulation rules. To understand the role of FUT8 in the glycosylation of CHO cells, we generated a FUT8 knock-out CHO cell line and performed a large-scale glycoproteomics to characterize the FUT8KO and wild-type (WT) CHO cells. The glycopeptides were enriched by hydrophilic chromatography and fractionated 25 fractions by bRPLC followed by analysis using high-resolution liquid chromatography mass spectrometry (LC-MS). A total of 7,127 unique N-linked glycosite-containing intact glycopeptides (IGPs), 928 glycosites, and 442 glycoproteins were identified from FUT8KO and WT CHO cells. Moreover, 28.62% in 442 identified glycoproteins and 26.69% in 928 identified glycosites were significantly changed in the FUT8KO CHO compared to wild-type CHO cells. The relative abundance of all the three N-glycan types (high-mannose, hybrid, and complex) was determined in FUT8KO comparing to wild-type CHO cells. Furthermore, a decrease in fucosylation content was observed in FUT8KO cells, in which core-fucosylated glycans almost disappeared as an effect of FUT8 gene knockout. Meantime, a total of 51 glycosylation-related enzymes were also quantified in these two cell types and 16 of them were significantly altered in the FUT8KO cells, in which sialyltransferases and glucosyltransferases were sharply decreased. These glycoproteomic results revealed that the knock-out of FUT8 not only influenced the core-fucosylation of proteins but also altered other glycosylation synthesis processes and changed the relative abundance of protein glycosylation.

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Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia

International Journal of Molecular Sciences ◽

10.3390/ijms22105218 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5218

Author(s):

Tomu Kamijo ◽

Takahiro Kaido ◽

Masahiro Yoda ◽

Shinpei Arai ◽

Kazuyoshi Yamauchi ◽

...

Keyword(s):

Cell Lines ◽

Cho Cells ◽

Fibrinogen Level ◽

Culture Media ◽

Chinese Hamster ◽

Plasma Fibrinogen ◽

Enzyme Linked Immunosorbent Assay ◽

Cho Cell ◽

Accelerated Degradation ◽

Heterozygous Patient

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients’ plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and “D:D” interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient’s hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

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Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

Molecular and Cellular Biology ◽

10.1128/mcb.4.1.173 ◽

1984 ◽

Vol 4 (1) ◽

pp. 173-180 ◽

Cited By ~ 32

Author(s):

S W Stanfield ◽

D R Helinski

Keyword(s):

Chinese Hamster Ovary ◽

Cho Cells ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Single Copy ◽

Chromosomal Dna ◽

Cho Cell ◽

Ovary Cells ◽

Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

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