frequency and higher cytotoxicity against an alkylating agent, N-methyl -N9-nitro-N-nitrosoguanidine (MNNG) than the original Nalm-6 in the presence of an inhibitor of O6-methylguanine-methyltransferase, i.e., O6-benzylguanine (O6-BG). Furthermore, we revealed that the expression of MSH2 had no effects on gene targeting efficiency. It suggests that the lack of mismatch repair functions is not a cause of high gene targeting of this cell line and also that efficient manipulation of genome is possible in the cells expressing MSH2/MSH6. Nalm-6-MSH+ is useful for detailed analyses of functions of genes in responses to DNA damaging agents in human cells.were purchased from Sigma-Aldrich, Wak.And enhance local mutagenic-NHEJ and HR, 1516647 appears a good candidate to overcome the limitation [4,5]. Nevertheless, design and selection of the zinc-finger domains that recognize unique sites of the chromosome are not easily predictable in silico. Hence, establishment of ZFNs often requires in vivo experiments to ensure that the designed enzymes do not introduce double-strand breaks at offtarget sites. Ready-to-use ZFNs are commercially available, butnumbers of genes that can be manipulated by the commercial ZFNs are still limited and made-to-order commercial ZFNs are costly. Interestingly, several human cell lines possess exceptionally high gene targeting efficiency. Nalm-6, a pre-B acute lymphoblastic leukemia cell line, is one of them and the ratio between homologous recombinants and random integrants, i.e., targeting efficiency, is reported 1?0 [6?0]. The efficiency might be comparable to that of chicken B-lymphocyte DT40, which is frequently used to examine functions of various genes involved in DNA repair, recombination and translesion DNA synthesis (TLS) [11]. In addition, Nalm-6 has a stable near-diploid karyotype with normal p53 status. Genes involved in double-strand break repair have been knocked out in this cell line [6]. However, Nalm-6 lacks the expression of MSH2, a component of mismatch repair proteins [6,12]. MSH2 forms two distinct heterodimers, i.e., MutSa (MSH2/MSH6) and MutSb (MSH2/ MSH3) in 
human cells, both of which play a critical role in recognition of mismatch bases in DNA and initiation of repair [13]. MutSa recognizes base-base mismatches and deletion/ insertion of one or two bases, 15481974 while MutSb preferentially recognizes larger insertion/deletion mispairs [14]. Defect of MSH2 leads to genome instability and extremely high spontaneous mutation frequency [15]. In addition to mismatch bases, bothEstablishment of Human Cell Line Nalm-6-MSH+MutS complexes are able to bind to a wide variety of lesions in DNA, e.g., alkylated bases in DNA, oxidative DNA damage [16], UV photoproducts [17] and DNA-crosslinks [18], suggesting that the complexes may act as a general sensor of DNA damage, which initiates downstream cellular responses such as apoptosis [19]. Therefore, cells deficient in MSH2 may exhibit abnormal responses against DNA damaging agents in comparison to cells with the repair functions. In this study, we analyzed the cause of deficiency of MSH2 expression and restored the expression of MSH2 in Nalm-6. The restoration led to stable expression of MSH6, which makes a complex with MSH2. In Nalm-6 cells, the MSH6 gene is intact but the protein is poorly expressed because of the lack of MSH2 expression [6,20]. The Nalm-6 cells expressing MSH2/MSH6, which we call Nalm-6-MSH+ hereafter, displayed substantially lower spontaneous mutation frequency and higher cytotoxicity against an alkylating agent, N-methyl -N9-nitro-N-nitrosoguanidine (MNNG) than the original Nalm-6 in the presence of an inhibitor of O6-methylguanine-methyltransferase, i.e., O6-benzylguanine (O6-BG). Furthermore, we revealed that the expression of MSH2 had no effects on gene targeting efficiency. It suggests that the lack of mismatch repair functions is not a cause of high gene targeting of this cell line and also that efficient manipulation of genome is possible in the cells expressing MSH2/MSH6. Nalm-6-MSH+ is useful for detailed analyses of functions of genes in responses to DNA damaging agents in human cells.were purchased from Sigma-Aldrich, Wak.