PUBLICATION

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

Authors

Vierstraete, J., Willaert, A., Vermassen, P., Coucke, P.J., Vral, A., Claes, K.B.M.

ID

ZDB-PUB-171204-20

Date

2017

Source

Scientific Reports 7: 16518 (Journal)

Registered Authors

Coucke, Paul, Willaert, Andy

Keywords

none

MeSH Terms

Genetic Testing*
BRCA2 Protein/deficiency*
Homologous Recombination*/radiation effects
Embryo, Nonmammalian
Radiation, Ionizing
Rad51 Recombinase/metabolism
Genotyping Techniques
Animals
Immunohistochemistry
Animals, Genetically Modified
Gene Knockdown Techniques
Zebrafish Proteins/deficiency*
Zebrafish/genetics*

(all 13)

PubMed

29184099 Full text @ Sci. Rep.

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.

Genes / Markers

Figures

Show all Figures

Expression

Gene	Antibody	Fish	Conditions	Stage	Qualifier	Anatomy	Assay	Figure
mCherry		oki011Tg	x-ray	Protruding-mouth		intestine cell	IHC	Fig. 4

Phenotype

Fish	Conditions	Stage	Phenotype	Figure
WT	x-ray	Protruding-mouth	intestine cell Ab10-h2afx labeling increased amount, abnormal	Fig. 5
WT	x-ray	Protruding-mouth	intestine cell Ab4-rad51 labeling increased amount, abnormal	Fig. 2
WT	x-ray	Protruding-mouth	intestine cell Ab4-rad51 labeling increased amount, abnormal	Fig. 5
WT	x-ray	Protruding-mouth	intestine cell Ab4-rad51 labeling increased amount, abnormal	Fig. 6
WT + MO1-brca2	x-ray	Protruding-mouth	intestine cell Ab10-h2afx labeling increased amount, abnormal	Fig. 5
WT + MO1-brca2	x-ray	Protruding-mouth	intestine cell Ab4-rad51 labeling amount, ameliorated	Fig. 5
brca2^cmg35/+	x-ray	Protruding-mouth	intestine cell Ab4-rad51 labeling amount, ameliorated	Fig. 6
brca2^cmg35/cmg35	x-ray	Protruding-mouth	intestine cell Ab4-rad51 labeling amount, ameliorated	Fig. 6
brca2^hg5/hg5	x-ray	Protruding-mouth	intestine cell Ab4-rad51 labeling amount, ameliorated	Fig. 6
brca2^{sa22682/sa22682}	x-ray	Protruding-mouth	intestine cell Ab4-rad51 labeling amount, ameliorated	Fig. 6

1 - 10 of 11

Show

Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
cmg35		Small Deletion	brca2
hg5		Point Mutation	brca2
oki011Tg	Tg(Xla.Eef1a1:gmnn-mCherry)	Transgenic Insertion
sa22682		Point Mutation	brca2

Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
brca2	CRISPR1-brca2	CRISPR
brca2	MO1-brca2	MRPHLNO

Fish

Fish
brca2^cmg35/cmg35
brca2^cmg35/+
brca2^hg5/hg5
brca2^{sa22682/sa22682}
oki011Tg
WT
WT + MO1-brca2

Antibodies

Name	Type	Isotypes	Host Organism
Ab4-rad51	polyclonal	IgG	Rabbit
Ab7-brdu	monoclonal		Mouse
Ab10-h2afx	polyclonal	IgG	Rabbit

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
mCherry	EFG	mCherry

Mapping

Vierstraete et al., 2017 ZDB-PUB-171204-20 PMID:29184099

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

Vierstraete et al., 2017

ZDB-PUB-171204-20
PMID:29184099