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UCE: A uracil excision (USER™)-based toolbox for transformation of cereals.

Authors :
Hebelstrup, Kim H.
Christiansen, Michael W.
Carciofi, Massimiliano
Tauris, Birgitte
Brinch-Pedersen, Henrik
Holm, Preben B.
Source :
Plant Methods; 2010, Vol. 6, p15-24, 10p
Publication Year :
2010

Abstract

Background: Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs. These problems are obviated by "The Uracil Specific Excision Reagent (USER™)" technology (New England Biolabs) which thus offers a new and very timeefficient method for engineering of big and complex plasmids. Results: By application of the USER™ system, we engineered a collection of binary vectors, termed UCE (USER cereal), ready for use in cloning of complex constructs into the T-DNA. A series of the vectors were tested and shown to perform successfully in Agrobacterium-mediated transformation of barley (Hordeum vulgare L.) as well as in biolistic transformation of endosperm cells conferring transient expression. Conclusions: The USER™ technology is very well suited for generating a toolbox of vectors for transformation and it opens an opportunity to engineer complex vectors, where several genetic elements of different origin are combined in a single cloning reaction. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
17464811
Volume :
6
Database :
Complementary Index
Journal :
Plant Methods
Publication Type :
Academic Journal
Accession number :
51990963
Full Text :
https://doi.org/10.1186/1746-4811-6-15