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Title: Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes

Abstract

Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured were measured in reduced serum with or without additives (1 ..mu..g/ml insulin, 3 x 10/sup -7/ M linoleic acid, 1 x 10/sup -8/ M H/sub 2/SeO/sub 3/) or bovine serum albumin (BSA, 1% wt/vol). With the additives and less than or equal to 0.5% fetal bovine serum (FBS), Ham's F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle's minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at the aprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxy-uridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBSmore » plus BSA; observed mutant frequencies induced by dimethylnitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.« less

Authors:
; ;
Publication Date:
Research Org.:
Univ. of California, Livermore
OSTI Identifier:
6355124
DOE Contract Number:
W-7405-ENG-48
Resource Type:
Journal Article
Resource Relation:
Journal Name: In Vitro; (United States); Journal Volume: 19:9
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CELL CULTURES; GROWTH; CLONING; EFFICIENCY; CULTURE MEDIA; COMPARATIVE EVALUATIONS; DRUGS; HAMSTERS; IMMUNE REACTIONS; MUTAGEN SCREENING; OVARIES; PHENOTYPE; SPONTANEOUS MUTATIONS; ANIMALS; BODY; FEMALE GENITALS; GONADS; MAMMALS; MUTATIONS; ORGANS; RODENTS; SCREENING; VERTEBRATES; 550300* - Cytology; 550400 - Genetics

Citation Formats

Carver, J.H., Salazar, E.P., and Knize, M.G.. Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes. United States: N. p., 1983. Web. doi:10.1007/BF02628961.
Carver, J.H., Salazar, E.P., & Knize, M.G.. Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes. United States. doi:10.1007/BF02628961.
Carver, J.H., Salazar, E.P., and Knize, M.G.. 1983. "Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes". United States. doi:10.1007/BF02628961.
@article{osti_6355124,
title = {Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes},
author = {Carver, J.H. and Salazar, E.P. and Knize, M.G.},
abstractNote = {Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured were measured in reduced serum with or without additives (1 ..mu..g/ml insulin, 3 x 10/sup -7/ M linoleic acid, 1 x 10/sup -8/ M H/sub 2/SeO/sub 3/) or bovine serum albumin (BSA, 1% wt/vol). With the additives and less than or equal to 0.5% fetal bovine serum (FBS), Ham's F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle's minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at the aprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxy-uridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethylnitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.},
doi = {10.1007/BF02628961},
journal = {In Vitro; (United States)},
number = ,
volume = 19:9,
place = {United States},
year = 1983,
month = 9
}
  • A Chinese hamster ovary auxotroph requiring glycine + adenosine + thymidine CHO AUXB1) was shown by us previously to lack several folylpolyglutamate synthetase (FPGS) type activities. Two revertants of AUXB1 have been isolated and found to contain altered forms of this enzyme. The revertant enzymes are more sensitive to heat inactivation than the parent CHO enzyme. ATP and MgCl/sub 2/ protect both revertant and parent CHO FPGS against rapid heat denaturation at pH 9.0, but not at pH 7.4. A genetically related auxotroph (CHO AUXB3) contains one-fifth the parent amount of FPGS. Unlike the FPGS from parent CHO and amore » genetically unrelated mutant requiring only glycine (CHO AUXB2), the AUXB3 enzyme specifically lacks tetrahydropteroyltetra(U-/sup 14/C)glutamate synthetase activity. These findings and polyethylene glycol fusion data with AUXB2 indicate that AUXB1 and AUXB3 each carry a mutation in the structural gene for a CHO FPGS that catalyzes tetrahydropteroyldi- as well as tetraglutamate formation. The altered form of FPGS in AUXB3 is responsible for its glycine + adenosine auxotrophy under standard culture conditions.« less
  • The growth inhibitions and parallel morphological changes due to three arsenic compounds (sodium arsenite, arsenate, and dimethylarsinate) are examined and compared systematically for the first time in cultured mammalian cells. The authors used suspension-adapted Chinese hamster ovary (CHO-S) cells and cultured them under low and high folate conditions to assess whether arsenic toxicity is related to 1-carbon metabolism. The concentration of arsenite required to produce a 50% growth rate inhibition is increased from 4 ..mu..M to 22 ..mu..M when the culture medium folate level is raised from 0.2 ..mu..M (low folate) to 2.0 ..mu..M (high folate). 66 references, 8 figures,more » 1 table.« less
  • When Chinese hamster ovary (CHO) cells are shifted from medium which contains serum into serum-free medium, they complete one cell doubling and arrest in the G/sub 1/ phase of the cell cycle. During the first 72 h of arrest, there is little change in intracellular adenosine 3' : 5'-phosphate (cAMP) level, and the cells retain their usual epithelial-like morphology. After 96 h, the cAMP level doubles, the magnitude of the prostaglandin E/sub 1/-induced changes in cAMP increases threefold, and the cells convert from a rounded, epithelial-like shape to an elongated, fibroblast-like form. The fact that these biochemical and cellular transitionsmore » are subsequent to the growth arrest shows that the cAMP increase is not the cause of the growth arrest but is consistent with a role for cAMP in the control of cell morphology. In addition, these changes point to the importance of the G/sub 1/ phase for initiating cAMP-related events.« less
  • The authors have investigated DNA-mediated transfer of aminopterin resistance conferred by plasmid and UV resistance conferred by genomic DNA to the Chinese hamster ovary (CHO) cell line UV-135, a UV-sensitive mutant defective in nucleotide excision repair. Plasmid pSV2gpt-CaPO/sub 4/ coprecipitates induced aminopterin resistance with equal efficiency in the 6-thioguanine-resistant, aminopterin-sensitive, repair-proficient parental line AA8-4(tg-1) and in UV-135(tg-2). Genetic and molecular evidence for genomic DNA-mediated transformation of UV-135(tg-2) cells with a putative excision repair gene were obtained. The overall frequency of drug and UV resistance cotransformation was 8 X 10(8) per cell plated. This frequency was ca. 200- to 500-fold greatermore » than that expected from coincident but independent UVr reversion and plasmid gene transfer events. DNA transfer techniques with this CHO system will be useful for further analysis of the essential structural DNA sequences, gene cloning, and expression of functional excision repair genes.« less
  • Type I and type II scavenger receptors, which have been implicated in the development of atherosclerosis and other macrophage-associated functions, differ only by the presence in the type I receptor of an extracellular cysteine-rich C-terminal domain. Stable Chinese hamster ovary (CHO) cell transfectants expressing high levels of either the type I or type II bovine scavenger receptors have been generated. Type I and type II receptors in these cells mediated high-affinity saturable endocytosis of both {sup 125}I-labeled acetylated low density lipoprotein (LDL) and {sup 125}I-labeled oxidized LDL with the distinctive broad ligand specificity characteristic of scavenger receptors. After incubation formore » 2 days with acetylated LDL, the transfected cells accumulated oil red O-staining lipid droplets reminiscent of those in macrophage foam cells, whereas untransfected CHO cells did not. Thus, macrophage-specific gene products other than the scavenger receptor are not required for modified-LDL-induced intracellular lipid accumulation. In transfected cells, acetylated LDL efficiently competed for both its own endocytosis and that of oxidized LDL. This nonreciprocal cross competition suggests that these ligands may bind to nonidentical but interacting sites on a single receptor. Results were similar for transfectants expressing either type I or type II scavenger receptors. The nonreciprocal cross competition seen in the transfected CHO cells differs from that previously observed with cultured macrophages.« less