Design of a selectable reporter for the detection of mutations in mammalian simple repeat sequences.
To study the mutator phenotype characteristic of tumors showing widespread replication errors at simple DNA repeat sequences (RER+), we designed a selectable reporter system for the detection of such mutations in mammalian cells. A hygromycin B phosphotransferase gene was rendered out-of-frame by the insertion of a (CA)13 dinucleotide repeat tract immediately following the ATG start codon, and subcloned into a retroviral expression vector containing a G418 (neo) selectable marker. Following transduction of this construct into cultured cells, clonal neo+ cell lines were established and then tested for their ability to form colonies in hygromycin B-containing medium. Using this system, we found that the HCT116, LS174T and LS180 human colon carcinoma cell lines acquire hygromycin resistance (hygr) at a 100-fold higher frequency than the HT29, SW480, DLD-1 and HCT15 human colon carcinoma and NIH3T3 fibroblast cell lines, and at a 25-fold higher rate than the Rat 6 embyro fibroblast cell line. DNA sequence analysis indicated that frameshift mutations had occurred within the CA dinucleotide repeat tract in HCT116 cells that became hygr. Thus, the mutation rates at simple repeated sequences in mammalian cell lines can be readily determined and studied using this system.
Kahn
S M
SM
Columbia-Presbyterian Cancer Center, New York, NY, USA.
Klein
M G
MG
Jiang
W
W
Xing
W Q
WQ
Xu
D B
DB
Perucho
M
M
Weinstein
I B
IB
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003062:Codon; D003110:Colonic Neoplasms; D017931:DNA Primers; D004261:DNA Replication; D016368:Frameshift Mutation; D006801:Humans; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae; D014162:Transfection; D014407:Tumor Cells, Cultured
D003062:Codon; D017931:DNA Primers