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Volumn 277, Issue 5331, 1997, Pages 1523-1526

Conditional mutator phenotypes in hMSH2-deficient tumor cell lines

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; AUTOSOMAL DOMINANT DISORDER; COLON CANCER; GENE MUTATION; HUMAN; HUMAN CELL; PHENOTYPE; PRIORITY JOURNAL; TUMOR CELL LINE; TUMOR GROWTH;

EID: 0030817162     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5331.1523     Document Type: Article
Times cited : (82)

References (41)
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    • Most of the cell lines used in these experiments were obtained from the American Type Culture Collection. MRC-5 was obtained from the Imperial Cancer Research Fund Cell Production Facility. 2774 was the kind gift of T. Kunkel. All cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Mutation rates were determined by the Luria-Delbrück fluctuation test [S. E. Luria and M. Delbrück, Genetics 28, 491 (1943)]. Inocula containing 100 cells were grown for 2 weeks on 35-mm tissue culture wells. Colonies formed after this time were treated with trypsin and transferred to 100-mm dishes. After 5 days, the replica cultures were plated on 6-thioguanine (5 μg/ml) to determine mutation rate at the HPRT locus or on 1 μM ouabain to determine the rate of mutation to resistance to this drug. After 2 weeks, plates were stained and colonies larger than 50 cells were counted. The mutation rate was calculated by the method of the mean [R. L. Capizzi and J. W. Jameson, Mutat. Res. 17, 147 (1973)].
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    • Microsatellite instability was analyzed as in (20). DNA purified from cultures grown from single cells of the indicated lines was used to amplify microsatellite loci. Four loci were assayed: BAT26, which contains a mononucleotide run; D10S197 and D16S521, which are both dinucleotide repeats; and D7S1794, which contains a trinucleotide repeat. The frequency of instability presented in the text represents the fraction of subclones from each of the lines exhibiting novel alleles at the loci. Patterns of alleles at one of the microsatellite loci (D10S197) in single cell clones isolated from 2774 are presented in Fig. 1.
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    • We thank D. Carroll, B. Preston, and J. Roth for critically reading this manuscript. Supported by a National Cancer Institute grant (R01-CA-62244) to M.M. and an NIH training grant (T32-CA-09602) to B.R.


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