Abstract
Previously, we described a method for a recombinase-directed stacking of new DNA to an existing transgenic locus. Here, we describe how we can similarly stack DNA molecules in vitro and that the in vitro derived gene stack can be incorporated into an Agrobacterium transformation vector by in vitro recombination. After transfer to the chromosome by Agroinfection, the transgenic locus harbors a new target site that can be used for the subsequent in vivo stacking of new DNA. Alternatively, the in vitro derived gene stack has the potential to be integrated directly into the plant genome in vivo at a preexisting chromosomal target. Being able to stack DNA in vitro as well as in vivo, and with compatibility between the two systems, brings new flexibility for using the recombinase-mediated approach for transgene stacking.
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Acknowledgments
We thank Z. Han for pC35SCreB and pZH36. This work received support from Guangdong Province, China Talent Funds 2010 and MOST/Ministry of Agriculture Grant 2010ZX08010-001. Authors also affiliated with the Key laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement.
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Chen, W., Ow, D.W. (2016). Protocol for In Vitro Stacked Molecules Compatible with In Vivo Recombinase-Mediated Gene Stacking. In: Murata, M. (eds) Chromosome and Genomic Engineering in Plants. Methods in Molecular Biology, vol 1469. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4931-1_3
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DOI: https://doi.org/10.1007/978-1-4939-4931-1_3
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