Background Connections between environment and genes are critical elements for leading to cancers in individuals. (WT), building cell lines for the gene mutation assay thus, specifically TK+/- cells. Furthermore, we developed experimental circumstances to carry out chromosome aberration (CA) and sister chromatid exchange (SCE) assays with cells. Utilizing the WT TK+/- and KO TK+/- cells, we assayed genotoxicity of MMC. In the gene mutation assay, the cytotoxic and mutagenic sensitivities of KO TK+/- cells had been greater than those of WT TK+/- cells. MMC induced lack of heterozygosity (LOH), bottom set substitutions in CpG tandem and sites mutations in GpG sites in both cell lines. However, the frequencies of LOH and base substitutions at CpG sites were significantly higher in KO TK+/- cells than in WT TK+/- cells. MMC also induced CA and SCE in both cell lines. The KO TK+/- cells displayed higher sensitivity than that displayed by WT TK+/- cells in the SCE assay. Conclusions These results suggest that Pol is usually a modulating factor for the genotoxicity of MMC and also that the established cell lines are useful for evaluating the genotoxicity of chemicals K02288 inhibitor from multiple endpoints in different hereditary backgrounds of Pol . Electronic supplementary materials The online edition of this content (doi:10.1186/s41021-016-0067-3) contains supplementary materials, which is open to authorized users. in cells with changed Pol activity, leading to TK+/- cells. Furthermore, to gain understanding into chromosomal occasions, we developed experimental circumstances for chromosome aberration (CA) and sister chromatid exchange (SCE) assays. To judge the utility from the cell lines to research protective jobs of Pol with regards to genotoxicity, we open Pol wild-type (WT) TK +/- and KO TK+/- cells to MMC and looked into gene mutations and chromosomal harm. MMC is certainly a chemotherapeutic agent that induces monofunctional adducts, and intra- and inter-strand DNA crosslinks on the gene mutation assay in the TK+/- cells set up in this research because it is well K02288 inhibitor known that Pol can bypass BPDE-DNA adducts within an error-free way [11]. MMC was bought from Nacalai Tesque (Kyoto, Japan) and used as a test chemical because the chemotherapeutic agent induces not only bulky DNA adducts but also inter- and intra-DNA crosslinks [29, 30] which are not well-known the contribution of Pol in their repair processes. Dimethyl sulfoxide (DMSO) was obtained from Wako (Osaka, Japan) and used as a solvent control. Establishment of was constructed using MultiSite Gateway Three-Fragment Vector Construction Kit (Thermo Fisher Scientific, Waltham, MA, USA) as previously described [35]. The targeting vector K02288 inhibitor was linearized by gene mutation assay Established cell lines harboring the 0.05. Mutation spectrum analysis of using primers TK ex4 Fw and TK ex4 Rv2 to classify the mutants as LOH type, allele were approximately 100 bp longer than those of the functional allele due to residual sequence of the targeting vector around the site. For non-LOH mutants, total RNA was extracted using ISOGEN II (Nippon Gene, Tokyo, Japan) and then amplified with PrimeScript? OneStep RT-PCR Kit ver. 2 (TaKaRa, Shiga, Japan) using the primers TK c176 Fw and TK c983 Rv. Resulting cDNA sequences were analyzed with 3130 Avant Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) using primers TK cDNA seq K02288 inhibitor Fw1 and TK cDNA seq Fw2. In the case of RNA splicing mutants, to analyze mutations around the splicing donor or acceptor sequences, genomic DNA was also extracted using Gentra Puregene Cell Kit (QIAGEN, Hilden, Germany), and the locus was amplified by PCR using sets TK95 Fw and TK4795 Rv for exons 1C4 and TK11175 Fw and DUSP2 TK12940 Rv for exon 5 to 7. The amplified genomic DNA sequences were analyzed using primers TK c176 Fw for exons 1C2, gTK ex3 seq Fw.