UCE: A uracil excision (USERTM)-based toolbox for transformation of cereals

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  • Molekylær Genetik og Bioteknologi
  • Institut for Genetik og Bioteknologi

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 (USERTM)" technology (New England Biolabs) which thus offers a new and very time-efficient method for engineering of big and complex plasmids.

Results

By application of the USERTM 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 USERTM 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.


Udgivelsesdato: 10 June 2010
OriginalsprogEngelsk
TidsskriftPlant Methods
Vol/bind6
Nummer15
Sider (fra-til)1-10
Antal sider10
ISSN1746-4811
DOI
StatusUdgivet - 2010

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