Process development for a recombinant Chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system

2004 ◽  
Vol 88 (4) ◽  
pp. 437-450 ◽  
Author(s):  
Edwin P. Huang ◽  
Christopher P. Marquis ◽  
Peter P. Gray
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Process Development ◽  
Expression System ◽  
Chinese Hamster ◽  
Cho Cell ◽  
Cho Cell Line

Discrete electrophoretic products of mitochondrial protein synthesis in the Chinese hamster ovary cell line

1977 ◽  
Vol 55 (10) ◽  
pp. 1064-1074 ◽  
Author(s):  
R. W. Yatscoff ◽  
K. B. Freeman
Keyword(s):  
Protein Synthesis ◽  
Cell Line ◽  
Electrophoretic Mobility ◽  
Chinese Hamster Ovary ◽  
Mitochondrial Protein ◽  
Chinese Hamster ◽  
Mitochondrial Proteins ◽  
Mitochondrial Protein Synthesis ◽  
Cho Cell ◽  
Cho Cell Line

Mitochondrial proteins labelled with [35S]methionine for 1 h in whole Chinese hamster ovary (CHO) cells in the presence of cycloheximide or emetine, known inhibitors of cytosolic protein synthesis, have been enumerated and characterized by their electrophoretic mobility in sodium dodecyl sulfate slab gel electrophoresis. Ten distinct electrophoretic bands were observed. The components were relatively stable during a 2 h postlabelling period. The same 10 bands were also seen with the CHO cell line tsH1, labelled at 40 °C, a temperature at which cytosolic but not mitochondrial protein synthesis is inhibited in this cell line, and with isolated mitochondria labelled in the presence of cycloheximide. An 11th band was present when [3H]leucine but not [35S] methionine was used for labelling. The width of the major band suggested that it consists of two components making a total of at least 12 proteins synthesized in mitochondria. The molecular weights of these mitochondrial proteins ranged from 5000 to 50000 and there was a sixfold difference in the relative molar amounts synthesized in a 1-h period in the presence of [3H]leucine or [3SS] methionine.No differences in number or electrophoretic mobility of the mitochondrially synthesized proteins were found among the seven CHO cell lines examined. These results suggest the stability of the mitochondrial genome in the 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.

Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium

1985 ◽  
Vol 21 (10) ◽  
pp. 588-592 ◽  
Author(s):  
Francois Gasser ◽  
Philippe Mulsant ◽  
Michel Gillois
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Serum Free Medium ◽  
Free Medium ◽  
Cho Cell ◽  
Serum Free ◽  
Cho Cell Line

Replication timing in a single human chromosome 11 transferred into the Chinese hamster ovary (CHO) cell line

Gene ◽  
2012 ◽  
Vol 510 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Yoshihisa Watanabe ◽  
Yasuhiro Kazuki ◽  
Mitsuo Oshimura ◽  
Toshimichi Ikemura ◽  
Masato Maekawa
Keyword(s):  
Human Chromosome ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Replication Timing ◽  
Chromosome 11 ◽  
Cho Cell ◽  
Cho Cell Line

O-109 A first dose-response trial investigating the effects of adding choriogonadotropin beta to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Fernandez Sanchez ◽  
H Višnová ◽  
C Blockeel ◽  
A Pinborg ◽  
Y Khalaf ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Cell Line ◽  
Gnrh Agonist ◽  
Ovarian Stimulation ◽  
Randomised Trial ◽  
Chinese Hamster ◽  
Ongoing Pregnancy ◽  
Cho Cell ◽  
Ongoing Pregnancy Rate ◽  
Cho Cell Line

Abstract Study question Does addition of choriogonadotropin beta (CG beta) to follitropin delta increase the number of good-quality blastocysts following ovarian stimulation in a long GnRH agonist protocol? Summary answer At the doses investigated, the addition of CG beta reduced the number of intermediate follicles and decreased the number of oocytes and blastocysts. What is known already CG beta is a new recombinant hCG (rhCG) molecule expressed by a human cell line (PER.C6â) with a different glycosylation profile compared to urinary hCG or rhCG derived from a Chinese Hamster Ovary (CHO) cell-line. In the first-in-human trial, the CG beta pharmacokinetics were similar between men and women. In women, the area under the curve (AUC) and the peak serum concentration (Cmax) increased dose proportionally following single and multiple daily doses. In men, a single dose of CG beta provided higher exposure with a longer half-life and proportionately higher testosterone production than rhCG derived from a CHO cell line. Study design, size, duration Placebo-controlled, double-blind, randomised trial (RAINBOW) to explore the efficacy and safety of CG beta as add-on treatment to follitropin delta in women undergoing COS in a long GnRH agonist protocol. The primary endpoint was the number of good-quality blastocysts (grade 3 BB or higher, Gardner and Schoolcraft, 1999). Subjects were randomised to receive either placebo or 1, 2, 4, 8, or 12 µg CG beta added to the daily individualised follitropin delta dose during COS. Participants/materials, setting, methods In total 619 women (30-42 years) with AMH levels between 5 and 35 pmol/L were randomized in equal proportions to the six treatment groups. All subjects were treated with an individualised dose of follitropin delta determined based on AMH (Elecsys AMH Plus Immunoassay) and body weight. Triggering was performed when 3 follicles were ≥17 mm but no more than 25 follicles ≥12 mm were reached Main results and the role of chance The incidence of cycle cancellation (range 0%-2.9%), total follitropin delta dose (mean 112 µg) and duration of stimulation (mean 10 days) were similar across the groups. A reduced number of intermediate follicles (12 to 17 mm) and fewer oocytes (mean range 9.7 to 11.2) were observed for all doses of CG beta compared to the follitropin delta only group (mean 12.5). The mean number of goodquality blastocysts was 3.3 in the follitropin delta group and ranged between 2.1 and 3.0 across the CG beta groups. The incidence of transfer cancellation was higher in the 4, 8 and 12 µg group, mostly as no blastocyst was available for transfer. In the group receiving only follitropin delta, the ongoing pregnancy rate (10-11 weeks after transfer) was high i.e. 43% per started cycle vs 28-39% in CG beta groups and 49% per transfer vs 38-50% in the CG beta groups. In line with the number of collected oocytes, the OHSS incidence was overall lower following follitropin delta with CG beta than following follitropin delta only treatment. Regardless of the dose, CG beta was safe and well-tolerated with low risk of immunogenicity. Limitations, reasons for caution The effect of the unique glycosylation of CG beta and the associated potency implications in women were not known prior to this trial. Further studies will be needed to evaluate potentially lower doses of CG beta for this and/or different indications. Wider implications of the findings The high ongoing pregnancy rate in the follitropin delta group supports the use of individualised follitropin delta dosing in a long GnRH agonist protocol. The differential potency of CG beta may have impaired the growth of intermediate follicles with the investigated doses without affecting the ongoing pregnancy rates per transfer. Trial registration number NCT03564509

scholarly journals Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse-human immunoglobulin G1.

1994 ◽  
Vol 180 (3) ◽  
pp. 1087-1096 ◽  
Author(s):  
A Wright ◽  
S L Morrison
Keyword(s):  
Cell Line ◽  
Half Life ◽  
Gene Transfection ◽  
Chinese Hamster ◽  
Chimeric Mouse ◽  
Carbohydrate Structure ◽  
Antigen Specificity ◽  
Cho Cell ◽  
Cho Cell Line

Immunoglobulin G (IgG) molecules are glycosylated in CH2 at Asn297; the N-linked carbohydrates attached there have been shown to contribute to antibody (Ab) stability and various effector functions. The carbohydrate attached to the IgG constant region is a complex biantennary structure. Alterations in the structure of oligosaccharide have been associated with human diseases such as rheumatoid arthritis and osteoarthritis. To study the effects of altered carbohydrate structure on Ab effector function, we have used gene transfection techniques to produce mouse-human chimeric IgG1 Abs in the Chinese hamster ovary (CHO) cell line Lec 1, which is incapable of processing the high-mannose intermediate through the terminal glycosylation steps. We also produced IgG1 Abs in Pro-5, the wild-type CHO cell line that is the parent of Lec 1. The Pro-5-produced Ab (IgG1-Pro-5) was similar to IgG1-My 1, a myeloma-produced IgG1 Ab of the same specificity, in its biologic properties such as serum half-life, ability to effect complement-mediated cytolysis, and affinity for Fc gamma RI. Although the Lec 1-produced Ab, IgG1-Lec 1, was properly assembled and retained antigen specificity, it was incapable of complement-mediated hemolysis and was substantially deficient in complement consumption, C1q binding, and C1 activation. IgG1-Lec 1 also showed reduced but significant affinity for Fc gamma R1 receptors. The in vivo half-life of IgG1-Lec 1 was shorter than that of either the myeloma- or Pro-5-produced counterpart, with more being cleared during the alpha-phase and with more rapid clearance during the beta-phase. Clearance of IgG1-Lec 1 could be inhibited by the administration of yeast-derived mannan. Thus the uptake of IgG1-Lec 1 appears to be accelerated by the presence of terminally mannosylated oligosaccharide. Therefore, certain Ab functions as well as the in vivo fate of the protein are dramatically affected by altered carbohydrate structure. Expression of Igs in cell lines with defined glycosylation mutations is shown to be a useful technique for investigating the contribution of carbohydrate structure to Ab function.

scholarly journals Canine and Feline Parvoviruses Can Use Human or Feline Transferrin Receptors To Bind, Enter, and Infect Cells

2001 ◽  
Vol 75 (8) ◽  
pp. 3896-3902 ◽  
Author(s):  
John S. L. Parker ◽  
William J. Murphy ◽  
Dai Wang ◽  
Stephen J. O'Brien ◽  
Colin R. Parrish
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Canine Parvovirus ◽  
Endocytic Pathway ◽  
Transferrin Receptors ◽  
Hybrid Cells ◽  
Cho Cell ◽  
Viral Capsids ◽  
Feline Panleukopenia Virus

ABSTRACT Canine parvovirus (CPV) enters and infects cells by a dynamin-dependent, clathrin-mediated endocytic pathway, and viral capsids colocalize with transferrin in perinuclear vesicles of cells shortly after entry (J. S. L. Parker and C. R. Parrish, J. Virol. 74:1919–1930, 2000). Here we report that CPV and feline panleukopenia virus (FPV), a closely related parvovirus, bind to the human and feline transferrin receptors (TfRs) and use these receptors to enter and infect cells. Capsids did not detectably bind or enter quail QT35 cells or a Chinese hamster ovary (CHO) cell-derived cell line that lacks any TfR (TRVb cells). However, capsids bound and were endocytosed into QT35 cells and CHO-derived TRVb-1 cells that expressed the human TfR. TRVb-1 cells or TRVb cells transiently expressing the feline TfR were susceptible to infection by CPV and FPV, but the parental TRVb cells were not. We screened a panel of feline-mouse hybrid cells for susceptibility to FPV infection and found that only those cells that possessed feline chromosome C2 were susceptible. The feline TfR gene (TRFC) also mapped to feline chromosome C2. These data indicate that cell susceptibility for these viruses is determined by the TfR.

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