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Sci Rep. 2017 Nov 28;7(1):16518. doi: 10.1038/s41598-017-16725-3.

Accurate quantification of homologous recombination in zebrafish: brca2 deficiency as a paradigm.

Author information

1
Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium.
2
Department for Basic Medical Sciences, Ghent University, Ghent, Belgium.
3
Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium. Kathleen.Claes@UGent.be.

Abstract

Homologous Recombination (HR) repair is essential for repairing DNA double strand breaks (DSB) in dividing cells and preventing tumorigenesis. BRCA2 plays an important role in HR by recruiting the DNA recombinase RAD51 to the DSB. Despite being a popular model organism in genetic and cancer research, knowledge on the conservation of the HR pathway and function of zebrafish Brca2 is limited. To evaluate this, we developed a Rad51 foci assay in zebrafish embryos. We identified the zebrafish embryonic intestinal tissue as an ideal target for Rad51 immunostaining. After inducing DSB through irradiation, Rad51 foci were present in irradiated embryos but not in unirradiated controls. We present a method for accurate quantification of HR. Both morpholino-induced knockdown and knockout of Brca2 lead to almost complete absence of Rad51 foci in irradiated embryos. These findings indicate conserved function of Brca2 in zebrafish. Interestingly, a statistically significant decrease in Rad51 foci was observed in Brca2 heterozygous carriers compared to wild types, indicative of haploinsufficiency, a hypothesised cause of some tumours in patients with a germline BRCA2 mutation. In conclusion, we demonstrated the suitability of zebrafish as an excellent in vivo model system for studying the HR pathway and its functionality.

PMID:
29184099
PMCID:
PMC5705637
DOI:
10.1038/s41598-017-16725-3
[Indexed for MEDLINE]
Free PMC Article

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