Abstract
BackgroundCloning 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 time-efficient method for engineering of big and complex plasmids.ResultsBy 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.ConclusionsThe 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.
Highlights
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
Single or multiple PCR products with long 3' overhangs generated by the uracil specific excision reaction can be inserted into linearized vectors with a compatible USERTM cassette to form a stable plasmid, which can be used to transform E. coli without prior ligation (Fig. 1A)
This requires that a complementary long 3' overhang is generated from the USERTM cassette when the vector is linearized
Summary
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 offers a new and very timeefficient method for engineering of big and complex plasmids. As the two species are very closely related genetically, barley is often used as a model for wheat and many transformation constructs can be used interchangeably. Cloning has to be performed in a multiple-cloning site (MCS) with a limited amount of unique or dual represented endonuclease recognition
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