A team of Austrian researchers this week published in Nature a genome-wide library of Drosophila RNAi transgenes that conditionally inactivate gene function in specific tissues of the fly.
According to investigators from the Institute of Molecular Biotechnology of the Austrian Academy of Sciences and the Research Institute of Molecular Pathology, the library could potentially enable researchers to systemically analyze gene function in any tissue and at any stage of the Drosophila lifespan.
“Geneticists have traditionally sought to gain insight into complex biological processes through forward genetic screens,” the researchers wrote in Nature. “Mutations are generated at random, phenotypes of interest are scored, and the mutated gene is subsequently identified.”
While this approach has been very successful, it has its limitations, they note. Specifically, there are “inherent biases in mutagenesis techniques, the large numbers of mutants that must be analyzed, and the considerable effort that is still required to identify the relevant genetic lesions.”
Additionally, the majority of genes have multiple functions, and one gene’s function in a certain tissue can “preclude its recovery in screens focused on functions in other tissues,” especially when the gene is essential in the early stages of the organism’s development.
The development of RNAi technologies and the availability of annotated genome sequences have enabled gene function analyses by reverse genetics, but large-scale RNAi-based surveys of gene function in vivo are currently limited to C. elegans and S. mediterranea, the authors wrote. Additionally, in these organisms RNAi is systemic, making cell type-specific gene silencing difficult.
In Drosophila, however, RNAi is cell-autonomous, they noted. “If a genome-wide library of transgenic RNAi strains were available, it would thus be possible to conduct systematic RNAi screens targeted to specific cell types in the intact animal.”
To develop such a resource, the researchers constructed a library of 15,072 UAS-driven inverted repeat (UAS-IR) transgenes — representing 13,327 different genes or 97 percent of predicted protein-coding genes — by cloning short gene fragments as inverted repeats into a modified pUAST vector, according to the paper.
Validated UAS-IR constructs were then used to generate a library of about 22,270 transgenic RNAi strains, representing 13,251 RNAi constructs and 12,088 genes that account for 88 percent of predicted protein-coding genes.
“To assess the targeting potential of each RNAi construct, we conceptually diced the predicted hairpin RNA into all possible 19-mers because RNAi-mediated degradation of a target messenger RNA generally requires a perfect match of at least 19 nucleotides,” the researchers explained. “We then interrogated the predicted Drosophila transcriptome for all perfect matches to these 19-mers, in both the sense and antisense orientations.”
After testing the efficacy of the RNAi constructs, the researchers examined their tissue specificity.
“If a genome-wide library of transgenic RNAi strains were available, it would thus be possible to conduct systematic RNAi screens targeted to specific cell types in the intact animal.”
“We selected 50 lines that were lethal in combination with the Act5C-GAL4 driver and crossed each of them separately to GAL4 drivers that target gene interference to the wing, eye, or notum,” they wrote.
While about 30 percent of the lines were lethal with each of the drivers, “the majority of lines were viable with the tissue-specific drivers, and 25 [to] 35 percent resulted in specific morphological defects,” they noted. Different sets of lines produced phenotypes with different GAL4 drivers, such that 80 percent of the lines produced a lethal or visible phenotype with at least one driver.”
Additional experiments showed that transgenic RNAi was potent in neurons and muscles, which are “internal tissues that are difficult or impossible to target by classical genetics,” the researchers added.
All of the RNAi lines generated by the research team are publicly available and they estimate that over 60 percent of the lines trigger potent and specific gene silencing, the researchers wrote. “As many as 90 percent [of the lines] may function in combination with the appropriate drivers, assays, and RNAi-enhancing tools.”
They note that their library, like RNAi in general, is subject to “variable efficiency of gene knock-down and the inherent risk of off-targeting effects. Additionally, because our transgenes are inserted at random sites, some false negatives may be caused by poor transgene expression and some false positives by the misregulation of flanking endogenous genes.”
The researchers concluded that the transgenic RNAi library, despite its limitations, “offers a powerful alternative to classical forward genetic screens. Although mutagenesis screens will continue to be useful, particularly for early embryonic development, transgenic RNAi screens should be especially suitable for later stages and whenever tissue-specific gene disruption is required.”