TY - JOUR
T1 - Evaluation of homologous recombinational repair in chicken B lymphoma cell line, DT40.
AU - Kitao, Hiroyuki
AU - Hirano, Seiki
AU - Takata, Minoru
PY - 2011/10/4
Y1 - 2011/10/4
N2 - 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.
AB - 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.
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U2 - 10.1007/978-1-61779-129-1_17
DO - 10.1007/978-1-61779-129-1_17
M3 - Article
C2 - 21660701
AN - SCOPUS:80053306431
VL - 745
SP - 293
EP - 309
JO - Methods in Molecular Biology
JF - Methods in Molecular Biology
SN - 1064-3745
ER -