Selectable and inheritable gene silencing through RNA interference in the unicellular alga Chlamydomonas reinhardtii

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.