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

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.

Characterization of a CHO cell line resistant to killing by the hypoxic cell cytotoxin SR 4233

1992 ◽  
Vol 22 (4) ◽  
pp. 681-684 ◽  
Author(s):  
Amato J. Giaccia ◽  
Kim A. Biedermann ◽  
Liana M. Tosto ◽  
Andrew I. Minchinton ◽  
Mary S. Kovacs ◽  
...  
Keyword(s):  
Cell Line ◽  
Hypoxic Cell ◽  
Cho Cell ◽  
Cho Cell Line

Isolation and Characterization of a CHO Cell Line Expressing rhFSH in Low Protein and Protein-Free Media

1997 ◽  
pp. 669-674
Author(s):  
Carol Luchette ◽  
Frank Deer ◽  
Kim Gurnett ◽  
Judy Rosenthal ◽  
Erik Johnson ◽  
...  
Keyword(s):  
Cell Line ◽  
Cho Cell ◽  
Low Protein ◽  
Isolation And Characterization ◽  
Cho Cell Line ◽  
Free Media

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells

2011 ◽  
Vol 109 (4) ◽  
pp. 1007-1015 ◽  
Author(s):  
Lianchun Fan ◽  
Ibrahim Kadura ◽  
Lara E. Krebs ◽  
Christopher C. Hatfield ◽  
Margaret M. Shaw ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Cell Line ◽  
Gene Knockout ◽  
Cho Cell ◽  
Glutamine Synthetase Gene ◽  
Cho Cell Line ◽  
Cell Line Generation ◽  
Line Generation

Generation and Characterization of a Stable Soluble Guanylate Cyclase-Overexpressing CHO Cell Line

Nitric Oxide ◽  
1999 ◽  
Vol 3 (1) ◽  
pp. 55-66 ◽  
Author(s):  
Eva Maria Becker ◽  
Frank Wunder ◽  
Raimund Kast ◽  
Chantal Robyr ◽  
Markus Hoenicka ◽  
...  
Keyword(s):  
Cell Line ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Cho Cell ◽  
Cho Cell Line

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

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.

CHARACTERIZATION OF A CHO CELL LINE RESISTANT TO KILLING BY SR 4233

1991 ◽  
pp. 349
Author(s):  
M.S. KOVACS ◽  
A.J. GIACCIA ◽  
K.A. BIEDERMANN ◽  
L. TOSTO ◽  
A. MINCHINTON ◽  
...  
Keyword(s):  
Cell Line ◽  
Cho Cell ◽  
Cho Cell Line

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

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.

scholarly journals 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

1997 ◽  
Vol 321 (1) ◽  
pp. 157-163 ◽  
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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