Document Detail

Gene targeting in maize by somatic ectopic recombination.
MedLine Citation:
PMID:  23094946     Owner:  NLM     Status:  Publisher    
Low transformation efficiency and high background of non-targeted events are major constraints to gene targeting in plants. We demonstrate here applicability in maize of a system that reduces the constraint from transformation efficiency. The system requires regenerable transformants in which all of the following elements are stably integrated in the genome: (i) donor DNA with the gene of interest adjacent to sequence for repair of a defective selectable marker, (ii) sequence encoding a rare-cutting endonuclease such as I-SceI, (iii) a target locus (TL) comprising the defective selectable marker and I-SceI cleavage site. Typically, this requires additional markers for the integration of the donor and target sequences, which may be assembled through cross-pollination of separate transformants. Inducible expression of I-SceI then cleaves the TL and facilitates homologous recombination, which is assayed by selection for the repaired marker. We used bar and gfp markers to identify assembled transformants, a dexamethasone-inducible I-SceI::GR protein, and selection for recombination events that restored an intact nptII. Applying this strategy to callus permitted the selection of recombination into the TL at a frequency of 0.085% per extracted immature embryo (29% of recombinants). Our results also indicate that excision of the donor locus (DL) through the use of flanking I-SceI cleavage sites may be unnecessary, and a source of unwanted repair events at the DL. The system allows production, from each assembled transformant, of many cells that subsequently can be treated to induce gene targeting. This may facilitate gene targeting in plant species for which transformation efficiencies are otherwise limiting.
Ayhan Ayar; Sophie Wehrkamp-Richter; Jean-Baptiste Laffaire; Samuel Le Goff; Julien Levy; Sandrine Chaignon; Hajer Salmi; Alexandra Lepicard; Christophe Sallaud; Maria E Gallego; Charles I White; Wyatt Paul
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-10-24
Journal Detail:
Title:  Plant biotechnology journal     Volume:  -     ISSN:  1467-7652     ISO Abbreviation:  Plant Biotechnol. J.     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-25     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101201889     Medline TA:  Plant Biotechnol J     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
© 2012 Biogemma Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.
Biogemma, Centre de Recherche de Chappes, Chappes, France; CNRS UMR 6293, Clermont Université INSERM U1103, Aubière, France.
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