Production of Chimeric Recombinant Single Domain Antibody—Green Fluorescent Fusion Protein in Chinese Hamster Ovary Cells

Hybridoma ◽  
2007 ◽  
Vol 26 (1) ◽  
pp. 1-9 ◽  
Author(s):  
M. Rajabi Bazl ◽  
M.J. Rasaee ◽  
M. Foruzandeh ◽  
A. Rahimpour ◽  
J. Kiani ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Ovary Cells ◽  
Domain Antibody ◽  
Green Fluorescent

scholarly journals Validation and identification of reference genes in Chinese hamster ovary cells for Fc-fusion protein production

2020 ◽  
Vol 245 (8) ◽  
pp. 690-702
Author(s):  
Xiaonan Ma ◽  
Ling Zhang ◽  
Luming Zhang ◽  
Chenglong Wang ◽  
Xiaorui Guo ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Chinese Hamster Ovary ◽  
Reference Genes ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Expression Stability ◽  
Fed Batch ◽  
Ovary Cells ◽  
Fc Fusion Protein

Chinese hamster ovary cells are the predominant cell lines used for bio-therapeutic production. Real-time quantitative PCR (RT-qPCR) and transcriptomics are powerful tools to understand and optimize the Chinese hamster ovary cells for higher productivity or better control of product qualities. Reliable reference genes, which were proved to be experiment-specific, are critical yardsticks. In this study, we compared expression stability of 20 candidate reference genes at mRNA level, including commonly used housekeeping genes, previous literature reported genes in Chinese hamster ovary cells producing an intact antibody, and new candidates suggested by our RNA-seq transcriptomic database, in RT-qPCR reactions in Fc-fusion protein-producing Chinese hamster ovary cells with various productivity during long-term cultivation and fed-batch cultures at 26 different conditions. geNorm, NormFinder, BestKeeper, and ΔCt programs and methods were utilized to analyze the gene expression stability and gave an overall ranking. Akr1a1, Gpx1, and Aprt in long-term cultivation and Akr1a1, Rps16 in fed-batch culture, which have not been reported previously, exhibited the highest stability of gene expression, while Pabpn1, Hirip3, and Actb in both sets of experiments together with Atp5f1 in long-term passage process showed the weakest stability. The results were then validated using GLP1-Fc (Glucagon-like peptide-1 Fc fusion protein) gene as the target with determined expression level which were doubly confirmed by both absolute RT-qPCR and confocal microscopy. These new references should be considered for the investigations on Chinese hamster ovary cells in related research. Impact statement In order to reveal potential genotype-phenotype relationship, RT-qPCR reactions are frequently applied which require validated and reliable reference genes. With the investigation on long-term passage and fed-batch cultivation of CHO cells producing an Fc-fusion protein, four new reference genes–Akr1a1, Gpx1, Aprt, and Rps16, were identified from 20 candidates with the aid of geNorm, NormFinder, BestKeeper, and ΔCt programs and methods. This article provided more verified options in reference gene selection in related research on CHO cells.

Hands-on experiments on glycemia regulation and type 1 diabetes

2015 ◽  
Vol 39 (3) ◽  
pp. 232-239 ◽  
Author(s):  
M. Mingueneau ◽  
A. Chaix ◽  
N. Scotti ◽  
J. Chaix ◽  
A. Reynders ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Green Fluorescent Protein ◽  
Chinese Hamster Ovary ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Pancreatic Cells ◽  
Green Fluorescent

In the present article, we describe a 3-day experimental workshop on glycemia regulation and type 1 diabetes that engages students in open-ended investigations and guided experiments leading to results that are not already known to them. After an initial questioning phase during which students observe PowerPoint slides depicting the glycemia (blood glucose levels) of individuals in various situations, students design, execute, and interpret experiments to address one of the following questions: 1) Which criteria must an animal model of diabetes fulfill? 2) How do pancreatic cells maintain glycemia constant? and 3) Is there a way to produce an insulin protein similar to the one released by human pancreatic cells? Students then 1) measure glycemia and glycosuria in control mice and in a mouse model of type 1 diabetes (Alloxan-treated mice), 2) measure the release of insulin by pancreatic β-cells (INS-1 cell line) in response to different concentrations of glucose in the extracellular medium, and 3) transfect Chinese hamster ovary cells with a plasmid coding for green fluorescent protein, observe green fluorescent protein fluorescence of some of the transfected Chinese hamster ovary cells under the microscope, and observe the characteristics of human insulin protein and its three-dimensional conformation using RASMOL software. At the end of the experimental session, students make posters and present their work to researchers. Back at school, they may also present their work to their colleagues.

scholarly journals Interferon γ Receptor Extracellular Domain Expressed as IgG Fusion Protein in Chinese Hamster Ovary Cells

1995 ◽  
Vol 270 (8) ◽  
pp. 3958-3964 ◽  
Author(s):  
Michael Fountoulakis ◽  
Cecilia Mesa ◽  
Georg Schmid ◽  
Reiner Gentz ◽  
Michael Manneberg ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Extracellular Domain ◽  
Interferon Γ ◽  
Ovary Cells
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