title: Characterisation of a new reporter system allowing high throughput in planta screening for recombination events before and after controlled DNA double strand break induction.
abstract: DNA double strand breaks (DSBs) are created either by DNA damaging reagents or in a programmed manner, for example during meiosis. Homologous recombination (HR) can be used to repair DSBs, a process vital both for cell survival and for genetic rearrangement during meiosis. In order to easily quantify this mechanism, a new HR reporter gene that is suitable for the detection of rare recombination events in high-throughput screens was developed in Arabidopsis thaliana. This reporter, pPNP, is composed of two mutated Pat genes and has also one restriction site for the meganuclease I-SceI. A functional Pat gene can be reconstituted by an HR event giving plants which are resistant to the herbicide glufosinate. The basal frequency of intra-chromosomal recombination is very low (10(-5)) and can be strongly increased by the expression of I-SceI which creates a DSB. Expression of I-SceI under the control of the 35S CaMV promoter dramatically increases HR frequency (10,000 fold); however the measured recombinant events are in majority somatic. In contrast only germinal recombination events were measured when the meganuclease was expressed from a floral-specific promoter. Finally, the reporter was used to test a dexamethasone inducible I-SceI which could produce up to 200x more HR events after induction. This novel inducible I-SceI should be useful in fundamental studies of the mechanism of repair of DSBs and for biotechnological applications.
authors.affiliation: UMR CNRS 6247 GRED, 24 avenue des Landais, 63177 Aubière, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Wehrkamp-Richter
authors.forename: Fabienne
authors.initials: F
authors.lastname: Degroote
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Laffaire
authors.forename: Wyatt
authors.initials: W
authors.lastname: Paul
authors.forename: Pascual
authors.initials: P
authors.lastname: Perez
authors.forename: Georges
authors.initials: G
authors.lastname: Picard
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics; D015152:Blotting, Northern; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D003907:Dexamethasone / Q000494:pharmacology*; D053209:Herbicide Resistance / Q000235:genetics; D006540:Herbicides / Q000494:pharmacology*; D008957:Models, Genetic; D011995:Recombination, Genetic / Q000235:genetics*
chemical_list: D029681:Arabidopsis Proteins; D006540:Herbicides; D003907:Dexamethasone