Why is the cell line CHO DG44 expression system based on DHFR amplification?
CHO DG44 cells are deficient in dihydrofolate reductase (dhfr), and therefore, cells transfected with the gene of interest and dhfr can be selected in medium without hypoxanthine and thymidine, which are essential for nucleotide synthesis via the salvage pathway.
What are CHO S cells?
CHO-S [suspension] A clonal isolate, derived from Chinese hamster ovary (CHO K1) cells. The CHO-S parental cell line was selected for growth and transfection efficiency. CHO-S cells are adapted to serum-free suspension culture.
How do CHO cells produce antibodies?
Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome.
What is DHFR selection system?
Gene amplification is achieved by drug resistance. In this case (DHFR-MTX selection system) CHO cells are subjected to increasing concentration of MTX making them resistance to MTX via gene amplification. In transfected cells the recombinant DNA consists of the GOI closely linked to the gene for DHFR.
How does MTX amplification work?
MTX binds to DHFR, thereby inhibiting the production of tetrahydrofolate. With insufficient levels of DHFR, cells are deprived of nucleoside precursors (hypoxanthine and thymidine) and die. Gene amplification is a technique used by molecular biologists.
What do CHO cells produce?
CHO cells are the most common mammalian cell line used for mass production of therapeutic proteins. They can produce recombinant protein on the scale of 3-10 grams per liter of culture.
What proteins do CHO cells produce?
In particular, CHO cells are used to produce recombinant therapeutic proteins and monoclonal antibodies (mAbs), such as adalimumab, bezlotoxumab, avelumab, dupilumab, durvalumab, ocrelizumab, and brodalumab (Kaneyoshi et al., 2019a; Henry et al., 2020).
What is DHFR and why is it important?
Dihydrofolate reductase is a small enzyme that plays a supporting role, but an essential role, in the building of DNA and other processes. It manages the state of folate, a snaky organic molecule that shuttles carbon atoms to enzymes that need them in their reactions.
Why is methotrexate used in cell culture?
Low concentrations of MTX are then used to amplify the transfected genes for increased protein expression. A single cell cloning protocol is also described. This can be used after each stage of MTX amplification to isolate high-expressing clones that are also consistent producers over longer culture periods.
How does methotrexate interfere with DHFR?
The inhibition of DHFR by folate antagonists (methotrexate) results in a deficiency in the cellular pools of thymidylate and purines and thus in a decrease in nucleic acid synthesis. Therefore, methotrexate interferes with DNA synthesis, repair, and cellular replication.
Why are CHO cells used for protein production?
The Advantages of CHO Cells Their tolerance to variations in pH, oxygen levels, temperature or pressure make them the ideal cell for large-scale culture. High recombinant protein yields and specific productivity. Thanks to genetic optimization, protein yields of 3-10 grams per liter of cell culture.
What is the CHO DG44 cells and media kit?
The CHO DG44 Cells (cGMP banked) and Media Kit is optimized for the growth of dihydrofolate reductase deficient (DHFR-) Chinese Hamster Ovary (CHO) cells and expression of recombinant proteins in suspension culture. The kit provides:
What is CD DG44 Culture Media?
GIBCO® CD DG44 Medium—a chemically defined, protein-free medium specifically designed to enable optimal growth and expression of DG44 cells in suspension culture. CHO DG44 cells (cGMP banked) —Parental CHO DG44 cells have been produced, banked and tested to meet cGMP quality standards.
Why is DG44 the dominant mammalian host for recombinant protein manufacturing?
DOI: 10.1016/j.bbrc.2005.12.111 Abstract The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system.
Can CHO DG44 cells produce anti-influenza virus monoclonal antibody?
Generation of a serum free CHO DG44 cell line stably producing a broadly protective anti-influenza virus monoclonal antibody Veronika Chromikova, RolesData curation, Investigation, Methodology, Supervision, Visualization, Writing – original draft, Writing – review & editing