Abstract
MultiSite Gateway cloning techniques based on homologous recombination facilitate the combinatorial assembly of basic genetic pieces (i.e., promoters, CDS, and terminators) into gene expression or gene silencing cassettes. pENFRUIT is a collection of MultiSite Triple Gateway Entry vectors dedicated to genetic engineering in fruits. It comprises a number of fruit-operating promoters as well as C-terminal tags adapted to the Gateway standard. In this way, flanking regulatory/labeling sequences can be easily Gateway-assembled with a given gene of interest for its ectopic expression or silencing in fruits. The resulting gene constructs can be analyzed in stable transgenic plants or in transient expression assays, the latter allowing fast testing of the increasing number of combinations arising from MultiSite methodology. A detailed description of the use of MultiSite cloning methodology for the assembly of pENFRUIT elements is presented.
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Acknowledgments
We wish to thank Paco Timoner for his help in the improvement of tomato agroinjection technique and his good advice, Asun Fernandez for sharing information on tomato introns, and Rafael Martínez for skilled nursing of tomato plants. This work was supported by the Spanish Ministry of Science and Education (Ramón y Cajal program, BIO2010-15384 and BIO2008-03434 projects), the European Commission (program I3P and EU-SOL project), and Fundación Genoma España (ESP-SOL project).
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Estornell, L.H., Granell, A., Orzaez, D. (2012). Exploiting MultiSite Gateway and pENFRUIT Plasmid Collection for Fruit Genetic Engineering. In: Dunwell, J., Wetten, A. (eds) Transgenic Plants. Methods in Molecular Biology, vol 847. Humana Press. https://doi.org/10.1007/978-1-61779-558-9_28
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DOI: https://doi.org/10.1007/978-1-61779-558-9_28
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