Abstract
RNA interference (RNAi) is a conserved mechanism in a wide range of eukaryotes. Introduction of synthetic dsRNA could specifically target suppression of a gene or could result in off-target silencing of another gene due to sequence similarity. To verify if the observed phenotype in an RNAi transgenic line is due to silencing of a specific gene or if it is due to another nontarget gene, a synthetic gene complementation approach could be used. Synthetic gene complementation described in this method uses the technology of synthesizing a variant of a native gene (used in RNAi silencing) to maximize the difference in DNA sequences while coding for the exact same amino acids as the original native gene. This is achieved through the use of alternate codons. The new variant gene is expressed in the original RNAi transgenic lines and analyzed for complementation of the RNAi phenotype. Complementation of the RNAi-induced phenotype will indicate gene-specific silencing and not off-target silencing.
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Acknowledgment
This work was supported by funds from the National Science Foundation and was carried out in the laboratory of Dr. Daniel Klessig. The author would like to thank Dr. N. H. Chua for providing the estradiol-inducible pER8 plant transformation vector; Dr. Peter Waterhouse, CSIRO, for pHANNIBAL gene silencing vector; and DNA 2.0, California, for designing and synthesizing syn and syn 2 SABP2.
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Kumar, D. (2015). Synthetic Gene Complementation to Determine Off-Target Silencing. In: Mysore, K., Senthil-Kumar, M. (eds) Plant Gene Silencing. Methods in Molecular Biology, vol 1287. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2453-0_21
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DOI: https://doi.org/10.1007/978-1-4939-2453-0_21
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