Homologous recombination (HR) is a mode of double-strand break (DSB) repair required for cell viability in vertebrate cells. Targeted integration of homologous DNA fragment by HR is usually a very rare event in vertebrate cells; however, in chicken B lymphoma cell line DT40, the ratio of targeted to random integration is extremely high. Although the underlying mechanism of this phenotype is not fully understood, DT40 has been utilized as a model cell line for a number of genetic analyses. Here we describe three assays for evaluating homologous recombinational repair (HRR) using DT40 as a model system, measuring gene-targeting frequency, analyzing HRR process of single DSB induced by yeast homing endonuclease I-SceI, and measuring sister chromatid exchange frequency. Combined with generation of gene-disrupted DT40 mutant cell line, these assays have been highly useful to investigate the mechanisms in HRR. Using these techniques, a role of HRR of not only Rad52 epistasis group genes but also genes whose mutation cause hereditary cancer syndrome, such as Fanconi anemia, has been established.
All Science Journal Classification (ASJC) codes
- Molecular Biology