Generation of Highly Productive Chinese Hamster Ovary Cells by Application of Metabolic Pool Selection During Cell Line Development

2019 ◽  
Vol 7 (5) ◽  
pp. 355-367
Author(s):  
Michael Thiele ◽  
Norbert Arnold ◽  
Axel J. Scheidig ◽  
Karsten Winkler
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Line Development ◽  
Ovary Cells ◽  
Metabolic Pool

scholarly journals Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin.

1981 ◽  
Vol 1 (6) ◽  
pp. 552-559 ◽  
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protein Synthesis ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Diphtheria Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Pseudomonas Aeruginosa Exotoxin

Nigericin and monensin, ionophores for Na+ and K+, have been found to enhance the cytotoxicities of abrin, ricin, and Pseudomonas aeruginosa exotoxin A in Chinese hamster ovary (CHO) cells. They do not affect the cytotoxicity of diphtheria toxin in the same cell line. Maximal sensitization of the CHO cells toward ricin and Pseudomonas toxin requires preculture of CHO cells in the presence of nigericin. Inhibition of protein synthesis in CHO cells by ricin or Pseudomonas toxin is also enhanced by preculture of CHO cells in the presence of nigericin. These results suggest a common step in the intoxication process of ricin and Pseudomonas toxin, the rate of which is facilitated by pretreatment with nigericin. This step is, however, not shared by the intoxication of CHO cells with diphtheria toxin.

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.

scholarly journals High-level stable expression of recombinant 5-HT1A 5-hydroxytryptamine receptors in Chinese hamster ovary cells

1992 ◽  
Vol 285 (3) ◽  
pp. 933-938 ◽  
Author(s):  
A Newman-Tancredi ◽  
R Wootton ◽  
P G Strange
Keyword(s):  
Adenylate Cyclase ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Receptor Gene ◽  
Chinese Hamster ◽  
Receptor Expression ◽  
Ovary Cells ◽  
Recombinant Cell ◽  
High Level

The human 5-hydroxytryptamine 5-HT1A receptor gene was transfected into Chinese hamster ovary cells. A series of recombinant monoclonal cell lines expressing the receptor were isolated and the properties of one cell line that expressed receptors at a high level (2.8 pmol/mg) were studied in detail. In ligand binding assays with the selective 5-HT1A receptor agonist 2-(NN-di[3H]propylamino)-8-hydroxy-1,2,3,4-tetrahydronaphthalene ([3H]8-OH-DPAT) only a single class of saturable high-affinity binding sites was detected, with a pharmacological profile in competition experiments essentially identical to that of the 5-HT1A receptor of bovine hippocampus. [3H]8-OH-DPAT binding to the recombinant cell membranes was inhibited by GTP, showing that the receptors in the transfected cells couple to G-proteins. A series of 5-hydroxytryptamine agonists inhibited forskolin-stimulated adenylate cyclase activity in the cells and, despite the high level of receptor expression, their apparent efficacies were similar to those observed for inhibition of adenylate cyclase in brain. This recombinant cell line provides a complete model system for studying the 5-HT1A receptor and its transmembrane signalling system. The recombinant cells can also be grown in suspension culture for long periods but, whereas 5-HT1A receptor numbers and receptor regulation by guanine nucleotides are maintained in suspension-grown cells, the inhibition of adenylate cyclase by the 5-HT1A receptor is gradually lost.

Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin

1981 ◽  
Vol 1 (6) ◽  
pp. 552-559
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protein Synthesis ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Diphtheria Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Pseudomonas Aeruginosa Exotoxin

Nigericin and monensin, ionophores for Na+ and K+, have been found to enhance the cytotoxicities of abrin, ricin, and Pseudomonas aeruginosa exotoxin A in Chinese hamster ovary (CHO) cells. They do not affect the cytotoxicity of diphtheria toxin in the same cell line. Maximal sensitization of the CHO cells toward ricin and Pseudomonas toxin requires preculture of CHO cells in the presence of nigericin. Inhibition of protein synthesis in CHO cells by ricin or Pseudomonas toxin is also enhanced by preculture of CHO cells in the presence of nigericin. These results suggest a common step in the intoxication process of ricin and Pseudomonas toxin, the rate of which is facilitated by pretreatment with nigericin. This step is, however, not shared by the intoxication of CHO cells with diphtheria toxin.

scholarly journals Recombinant Thyroid Peroxidase-Specific Fab Converted to Immunoglobulin G (IgG) Molecules: Evidence for Thyroid Cell Damage by IgG1, but Not IgG4, Autoantibodies1

1997 ◽  
Vol 82 (3) ◽  
pp. 925-931 ◽  
Author(s):  
Jin Guo ◽  
Juan Carlos Jaume ◽  
Basil Rapoport ◽  
Sandra M. McLachlan
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Thyroid Peroxidase ◽  
51Cr Release ◽  
Thyroid Cells ◽  
Ovary Cells

Abstract A recombinant autoantibody Fab (SP1.4) to thyroid peroxidase (TPO), cloned from intrathyroidal B cell immunoglobulin genes, interacts with an epitope on TPO recognized by all patients with autoimmune thyroid disease. To compare the biological properties of IgG1 and IgG4 TPO autoantibodies, we converted Fab SP1.4 to full-length immunoglobulins. The SP1.4 heavy and κ light chain variable region genes, spliced by overlap PCR to a mammalian signal peptide, were transferred to expression vectors for human IgG1, IgG4, and κ L chains. Plasmids containing the IgG1 (or IgG4) heavy chain and the κ L chain were cotransfected into SP2/0 mouse myeloma cells. Cells secreting TPO autoantibodies were cloned, and IgG1-SP and IgG4-SP were affinity purified from medium using protein G. Their subclass specificities were confirmed by enzyme-linked immunosorbent assay and fluorometry after binding to Chinese hamster ovary cells expressing cell surface TPO. Further confirmation of SP1.4 Fab conversion to full-length molecules was the ability of protein A to precipitate IgG1-SP and IgG4-SP complexed to [125I]TPO. IgG1-SP1.4, IgG4-SP1.4, and Fab SP1.4 had similar high affinities for TPO (Kd = ∼2× 10−10 mol/L). Complexes of [125I]TPO and IgG1-SP (but not IgG4-SP) bound to peripheral blood mononuclear cells (PBMC), but not to a B cell line. Flow cytometry demonstrated Fc receptors FcγRI, FcγRII, and FcγRIII on PBMC, but only FcγRII on the B cell line. Together, these data indicate that IgG1-SP/TPO complexes bind to either FcγRI on monocytes or RIII on natural killer cells. In assays for antibody-dependent cytotoxicity using PBMC, 51Cr release was higher for thyroid cells preincubated with IgG1-SP (13.4%) than with IgG4-SP (2.5%) or with culture medium alone (−0.7%). No specific 51Cr release was observed when either fibroblasts or Chinese hamster ovary cells expressing cell surface TPO were used as target cells. In conclusion, a human TPO-specific Fab converted to IgG1, but not IgG4, can mediate cytotoxic effects on human thyroid cells in vitro. These observations support the clinical relevance of TPO autoantibody subclass distribution and emphasize the likelihood that, as opposed to being simple markers of thyroid damage, TPO autoantibodies may play a role in the induction of thyroid dysfunction in vivo.

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