Characterisation of a new reporter system allowing high throughput in planta screening for recombination events before and after controlled DNA double strand break induction.
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.
Wehrkamp-Richter
Sophie
S
UMR CNRS 6247 GRED, 24 avenue des Landais, 63177 Aubière, France.
Degroote
Fabienne
F
Laffaire
Jean-Baptiste
JB
Paul
Wyatt
W
Perez
Pascual
P
Picard
Georges
G
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*
D029681:Arabidopsis Proteins; D006540:Herbicides; D003907:Dexamethasone