Tetrahydrofolate increases suspension growth of dihydrofolate reductase-deficient chinese hamster ovary DG44 cells in chemically defined media

2016 ◽  
Vol 32 (6) ◽  
pp. 1539-1546
Author(s):  
Bong Gyun Kim ◽  
Hong Woo Park
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Chemically Defined Media ◽  
Defined Media

scholarly journals Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells

1989 ◽  
Vol 264 (30) ◽  
pp. 18005-18010 ◽  
Author(s):  
D C Thomas ◽  
D S Okumoto ◽  
A Sancar ◽  
V A Bohr
Keyword(s):  
Dna Repair ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Reductase Gene

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11-derived dihydrofolate reductase deficient host cell

2013 ◽  
Vol 29 (4) ◽  
pp. 980-985 ◽  
Author(s):  
Zhilan Hu ◽  
Donglin Guo ◽  
Shirley S.M. Yip ◽  
Dejin Zhan ◽  
Shahram Misaghi ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Stable Cell Line ◽  
Cell Line Development

Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells

1993 ◽  
Vol 13 (8) ◽  
pp. 5085-5098
Author(s):  
A M Carothers ◽  
G Urlaub ◽  
D Grunberger ◽  
L A Chasin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Consensus Sequence ◽  
Donor Site ◽  
Chinese Hamster ◽  
Base Substitution ◽  
Acceptor Site ◽  
Rnase Protection ◽  
Single Base

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1926-1935
Author(s):  
P J Mitchell ◽  
G Urlaub ◽  
L Chasin
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Splice Sites ◽  
Ovary Cells ◽  
Splicing Mutations ◽  
Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

scholarly journals Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line.

1981 ◽  
Vol 1 (12) ◽  
pp. 1069-1076 ◽  
Author(s):  
R J Kaufman ◽  
R T Schimke
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Ovary Cells ◽  
Methotrexate Resistance ◽  
Gene Copies ◽  
Methotrexate Concentration ◽  
Ovary Cell Line

During stepwise increases in the methotrexate concentration in culture medium, we selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). We studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells

1977 ◽  
Vol 55 (4) ◽  
pp. 445-452 ◽  
Author(s):  
Radhey S. Gupta ◽  
Wayne F. Flintoff ◽  
Louis Siminovitch
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Class I ◽  
Class Iii ◽  
Wild Type ◽  
Ovary Cells ◽  
I Cells ◽  
Resistant Cells

We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levels of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W. F., Davidson, S. V., and Siminovitch, L. (1976) Somatic Cell Genet. 2, 245–261). By selecting for increased resistance from these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In this report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and a class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.

Sequential Conformation Changes of Chinese Hamster Ovary Dihydrofolate Reductase at Its Active Sites

2005 ◽  
Vol 12 (5) ◽  
pp. 483-486
Author(s):  
Wen-he Zhang ◽  
San-bo Qin ◽  
Hong-jie Zhang ◽  
Zhi-yong Zhu
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Active Sites ◽  
Chinese Hamster ◽  
Conformation Changes

scholarly journals Expression and amplification of engineered mouse dihydrofolate reductase minigenes.

1983 ◽  
Vol 3 (2) ◽  
pp. 257-266 ◽  
Author(s):  
G F Crouse ◽  
R N McEwan ◽  
M L Pearson
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Cdna Clones ◽  
Coding Region ◽  
Chromosomal Dna ◽  
Lambda Phage ◽  
Ovary Cells

We constructed mouse dihydrofolate reductase (DHFR) minigenes (dhfr) that had 1.5 kilobases of 5' flanking sequences and contained either none or only one of the intervening sequences that are normally present in the coding region. They were greater than or equal to 3.2 kilobase long, about one-tenth the size of the corresponding chromosomal gene. Both of these minigenes complemented the DHFR deficiency in Chinese hamster ovary dhfr-1-cells at a high frequency after DNA-mediated gene transfer. The level of DHFR enzyme in various transfected clones varied over a 10-fold range but never was as high as in wild-type Chinese hamster ovary cells. In addition, the level of DHFR in primary transfectants did not vary directly with the copy number of the minigene, which ranged from fewer than five to several hundred per genome. The minigenes could be amplified to a level of over 2,000 copies per genome upon selection in methotrexate, a specific inhibitor of DHFR. In one case, the amplified minigenes were present in a tandem array; in two other cases, a rearranged minigene plasmid and its flanking chromosomal DNA sequence were amplified. Thus, the mouse dhfr minigenes could be transcribed, expressed, and amplified in Chinese hamster ovary cells, although the efficiency of expression was generally low. The key step in the construction of these minigenes was the generation in vivo of lambda phage recombinants by overlapping regions of homology between genomic and cDNA clones. The techniques used here for dhfr should be generally applicable to any gene, however large, and could be used to generate novel genes from members of multigene families.

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