Liposome-mediated transfer of YAC DNA to tobacco cells |
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| Authors: | Monique van Wordragen Roshani Shakya Ruud Verkerk Regis Peytavis Ab van Kammen Pim Zabel |
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| Institution: | (1) Department of Molecular Biology, Wageningen Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands;(2) Present address: ATO-DLO, PO-box 17, 6700 AA Wageningen, The Netherlands;(3) Present address: Friedrich Miescher Institut, P.O. Box 2543, CH-4002 Basel, Switzerland;(4) Present address: Department of Food Science, Wageningen Agricultural University, Bomenweg 2, 6703 HD Wageningen, The Netherlands |
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| Abstract: | This article describes a set of protocols—for retrofitting, transformation and purification—that together enable the delivery
of full-sized YAC-DNA to plant cells. To be able to equip YACs of interest with plant selectable markers, we have constructed
a retrofitting vector that carriesnptII anduidA. Furthermore, we established a transformation protocol for plant protoplasts that is sufficiently efficient to support transfer
of high-molecular-weight DNA. In this protocol lipofection is combined with PEG-mediated direct gene transfer. Large amounts
of purified DNA are necessary for lipofection. To obtain sufficient quantities of concentrated, purified YAC-DNA, we used
an optimized two-step, gel-purification method. Transient expression of a YAC-bornuidA demonstrates that both retrofitting vector and transformation protocol are effective. |
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| Keywords: | gel-purification Nicotiana tabacum SR1 retrofitting tobacco transformation yeast artificial chromosome |
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