Accurate quantification of homologous recombination in zebrafish : brca2 deficiency as a paradigm
Autor: | Andy Willaert, Anne Vral, Paul Coucke, Petra Vermassen, Jeroen Vierstraete, Kathleen Claes |
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Jazyk: | angličtina |
Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
Embryo Nonmammalian Genotyping Techniques ved/biology.organism_classification_rank.species genetic processes RAD51 lcsh:Medicine medicine.disease_cause DOUBLE-STRAND BREAKS Animals Genetically Modified 0302 clinical medicine Radiation Ionizing Recombinase Homologous Recombination lcsh:Science Zebrafish Gene knockdown Multidisciplinary INHIBITOR Immunohistochemistry CANCER GENOME Gene Knockdown Techniques 030220 oncology & carcinogenesis embryonic structures biological phenomena cell phenomena and immunity animal structures DNA damage EPITHELIUM Biology Article 03 medical and health sciences medicine TUMORIGENESIS Animals Genetic Testing Model organism BRCA2 Protein REPAIR ved/biology lcsh:R Biology and Life Sciences Zebrafish Proteins biology.organism_classification Molecular biology MODEL enzymes and coenzymes (carbohydrates) 030104 developmental biology DNA-DAMAGE FANCONI-ANEMIA lcsh:Q Rad51 Recombinase Homologous recombination Carcinogenesis |
Zdroj: | SCIENTIFIC REPORTS Scientific Reports, Vol 7, Iss 1, Pp 1-10 (2017) Scientific Reports |
ISSN: | 2045-2322 |
Popis: | 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. |
Databáze: | OpenAIRE |
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