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
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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