Abstract
Reverse genetic approaches have become invaluable tools to tap into the wealth of information provided by sequenced genomes. In 2007, sequencing of the Chlamydomonas reinhardtii genome was completed, and with this an increased demand for the development of reverse genetic strategies for gene analysis. In a variety of organisms, including Chlamydomonas, inverted repeat transgenes have been used to produce strains silenced for a specific gene due to the production of double stranded RNA (dsRNA). Here, we describe a tandem inverted repeat system designed to overcome some of the typical challenges that arise when transgenes are used to trigger gene silencing including the lack of a screenable phenotype, unpredictable levels of silencing, silencing of the transgene itself and thus loss of target gene silencing, and finally silencing of unintended genes (off-target genes). The described strategy allows selection of target gene silencing by inducing co-silencing of the target gene and a gene, MAA7, silencing of which produces a selectable RNAi-induced phenotype. This selection, therefore, precludes extensive molecular screening for transgenic strains exhibiting target gene silencing, and also ensures heritable silencing through many generations.
An erratum to this chapter is available at http://dx.doi.org/10.1007/978-1-61779-197-0_29
An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-61779-197-0_29
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Acknowledgments
Research in the van Dijk laboratory is supported by Grant Number P20 RR16469 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), Grant number 0940177 from the National Science Foundation (NSF) and Grant number EPS-1004094 from NSF.
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van Dijk, K., Sarkar, N. (2011). Selectable and Inheritable Gene Silencing through RNA Interference in the Unicellular Alga Chlamydomonas reinhardtii . In: Williams, J. (eds) Strain Engineering. Methods in Molecular Biology, vol 765. Humana Press. https://doi.org/10.1007/978-1-61779-197-0_28
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DOI: https://doi.org/10.1007/978-1-61779-197-0_28
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