CHO dhfr

Martiniuk, F., Chen, A., Donnabella, V. et al. (2000) Correction of glycogen storage disease type II by enzyme replacement with a recombinant human acid maltase produced by over-expression in a CHO-DHFR(neg) cell line. Biochemical and Biophysical Research Communications, 276 (3), 917-923. 

Suspension cultures can be achieved using F12 medium with 10% foetal bovine serum (FBS) and gassing the culture vessels continuously with 5% CO2. The doubling time under these conditions is typically 18 h versus 14 h for monolayer cultures. The growth requirements of CHO dhfr cells are hypoxanthine (or adenine), glycine and thymidine in addition to the usual requirement of CHO cells for proline. 

A WHO bank of CHO dhfr cells is deposited at the European Collection of Cell Cultures (ECACC) and permission can be obtained for their use in commercial production by a licence agreement through Professor Chasin, Department of Biological Sciences, Sherman Fairchild Centre for the Life Sciences, Columbia University, New York, NY 10027. The licence is subject to a one-time payment. 

CHO dhfr- Chinese hamster ovary cells Recombinant protein Urlaub et al. 

Two studies with discrepant results have been published on the pharmacologic effect of the Arg64 substitution in the /i3AR using recombinant expression. In CHO(dhfr-) cells, Strader and colleagues (86) found no differences in agonist... 

Although HKBll is not a dhfr (dihydrofolate reductase)-negative cell line, stable clones can be generated in a selection medium containing 100 nM MTX (methotrexate). Amplification of the gene cassette with increasing concentrations of MTX is possible comparable to the CHO dhfr system. 

Bohr, V.A., Smith, C.A., Okumoto, D.S., Hanawalt, P.C. (1985). DNA repair in an active gene removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 40, 359-369. 

Lucas, B.K., L.M. Giere, R.A. DeMarco, A. Shen, V. Chisholm, and C.W. Crowley. 1996. High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector. Nucleic Acids Res 24 1774-1779. 

Briefly, in the first step an expression plasmid containing the FVIII cDNA was co-transfected into dihydrofolate reductase (DHFR)-deficient CHO cells (DUKX-Bll) along with a plasmid expressing a DHFR-selectable marker to create the cell line lOAl. Methotrexate was then used to amplify FVIII expression. In the second step, it was decided to co-express vWF in the... 

Fig. 1.2 Generation of stable cell lines using DHFR-minus CHO cells. The example here uses co-transfection of restriction enzyme linearized plasmids. Clones appear 2-3 weeks after exposure to selective environmental conditions - here, culture of cells in media lacking hypoxanthine and thymidine.

To date, commercial antibody expression exclusively uses mammalian cell line expression. These mammalian cell lines have expression cassettes for the antibody heavy and light chain stably integrated into the host cell chromosome. The most commonly used cell lines are derived from Chinese hamster ovary (CHO) cells. About half the approved antibody therapeutics are made in CHO cell lines. The dihydrofolate reductase (dhfr) gene is used as a selectable marker owing to the development of CHO cell lines that are deficient for dhfr genes such as CHO DG44 and CHO... 

In a recent study [55], (III. 156), (III. 160), and the lO-CHO derivative (III. 163) were tested against DHFR from HCT-8 human colon tumour cells, and were found to have IC50 values of 0.05,0.35, and 0.009 pM, respectively, as compared with 0.0013 /rM for MTX and 0.00065 /rM for 5-methyl-5,8-dide-azaAMT (III. 148). An interesting feature of these results was the rather high potency of (III. 163), which was consistent with the data obtained against DHFR from L5178Y murine lymphoma cells by Dedhar et al. [47] and was reminiscent of the anti-DHFR activity reported a number of years ago for A °-formylfolate [56]. 

Besides, Tabuchi and Sugiyama [65] recently achieved maximum cell-specific mAB productivities greater than 100 pgcell" day with a DHFR-deficient CHO DUK-XBll cell line overexpressing the taurine transporter TAUT and the alanine aminotransferase 1 ALTl for amplifying the internal pyruvate and glutamate supply. 

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