Design of a selectable reporter for the detection of mutations in mammalian simple repeat sequences

To study the mutator phenotype characteristic of tumors showingwidespread replication errors at simple DNA repeat sequences(RER+), we designed a selectable reporter system for the detectionof such mutations in mammalian cells. A hygromycin B phosphotransferasegene was rendered out-of-frame by the insertion of a (CA)₁₃dinucleotide repeat tract immediately following the ATG startcodon, and subcloned into a retroviral expression vector containinga G418 (neo) selectable marker. Following transduction of thisconstruct into cultured cells, clonal neo+ cell lines were establishedand then tested for their ability to form colonies in hygromycinB-containing medium. Using this system, we found that the HCT116,LS174T and LS180 human colon carcinoma cell lines acquire hygromycinresistance (hyg^r) at a 100-fold higher frequency than the HT29,SW480, DLD-1 and HCT15 human colon carcinoma and NIH3T3 fibroblastcell lines, and at a 25-fold higher rate than the Rat 6 embyrofibroblast cell line. DNA sequence analysis indicated that frameshiftmutations had occurred within the CA dinucleotide repeat tractin HCT116 cells that became hyg^r. Thus, the mutation rates atsimple repeated sequences in mammalian cell lines can be readilydetermined and studied using this system.