Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas

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Abstract
RNA interference (RNAi), the double-stranded RNA (dsRNA) triggered post-transcriptional gene silencing, is becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of inverted repeat (IR) transgenes, has been achieved in protozoa, algae, fungi, plants, and metazoans. However, the level of gene silencing is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. This is a hindrance in functional genomics studies, where it is desirable to suppress target genes reliably to analyze unknown phenotypes. Consequently, we explored strategies for direct selection of effective transgenic RNAi lines in Chlamydomonas reinhardtii. We initially attempted to suppress expression of the Rubisco small subunit multigene family by placing an IR, homologous to the conserved coding sequence, in the 3′UTR of a transgene conferring resistance to bleomycin. However, this approach was fairly inefficient at inducing RNAi as many strains displayed defective transgene integration, resulting in partial or complete deletion of the IR, or low levels of dsRNA expression, presumably due to transcriptional silencing of the integrated IR transgenes. To overcome these problems we designed a system consisting of tandem IR transgenes that consistently triggered co-silencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase β subunit) and another gene of interest (encoding either Ku80, an RNA-binding protein, or a thioredoxin isoform). We anticipate that this approach will be useful for generating stable hypomorphic epi-mutants in high-throughput phenotypic screens.