ACGH analyses of DSB-induced LOH in Blm-deficient mESCs

ABSTRACT: Genome modification in mouse embryonic stem cells (mESCs) has provided the versatile platform to analyzing gene functions in mammalian cells. Bloom helicase-deficient mESCs showed elevated rate of loss of heterozygosity (LOH) through mitotic recombination. By utilizing this characteristic of Blm deficiency, we established an efficient method to introduce homozygous mutations into mESCs, which can be used for phenotype-driven recessive screen. It is generally thought that mitotic recombination is initiated by double-strand break. DNA repair machinery, however, possesses mechanisms that suppress deleterious crossovers between homologous chromosomes. Bloom helicase plays a central role to suppress such crossover; hence its deficiency causes elevated frequency of mitotic recombination. To directly answer if DSB can initiate mitotic recombination, we established a reporter cell line, in which the recognition site of I-SceI endonuclease was introduced in the Aprt gene in Blm-deficient mESCs. We used two types of I-SceI expression vectors; one expresses a mammalian codon-optimized I-SceI (ISceIo) and the other expresses I-SceIo fused with N-terminal 110-amino acids of Geminin (Gemi). Geminin-fused I-SceI can be stabilized only in lateS/G2 phase. After I-SceI transfection and subsequent selection, we isolated Aprt-deficient mutants. Half of the mutants showed LOH in the whole telomeric region from I-SceI site, but the other half showed local LOH near the I-SceI site. Further analyses suggested that large deletion was introduced and caused LOH, which was not seen in spontaneous LOH. We designed CGH probes in 300-bp resolution in 8Mb telomeric region of chr 8 (The Aprt gene locates 7Mb from the telomere) to map the deleted regions in these mutants.

ORGANISM(S): Mus musculus

SUBMITTER: Kosuke Yusa 

PROVIDER: E-MTAB-1116 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress