A team of researchers led by Harvard Medical School professor Norbert Perrimon has formally kicked off an effort to build a collection of transgenic RNAi lines for nearly half of all the genes within the Drosophila genome, RNAi News has learned.
Supported by a four-year grant from the National Institutes of Health worth roughly $2 million in direct costs, the Transgenic RNAi Resource Project, or TRiP, aims to create lines for 6,250 Drosophila genes, which will be transferred to and made available by the Bloomington Drosophila Stock Center at Indiana University.
Perrimon told RNAi News last week that he hopes to secure additional financing in the future to complete the resource.
The TRiP is an outgrowth of Harvard’s Drosophila RNAi Screening Center, which was established in 2003 to provide investigators with access to technology and expertise for their own screening experiments.
“People develop an assay in their own lab and come to our center for a few weeks or so and conduct the screen using the reagents and equipment we have,” Perrimon explained. “Over the past six years or so, we’ve done about 100 genome-wide RNAi screens.”
According to the DRSC, it helps researchers develop and optimize assays, conduct data and image analysis, and plan follow-up assays. Additionally, because reagent generation, staff support, and equipment at the center are grant-subsidized, it can be significantly less expensive for researchers to conduct screens at the facility than to do it at their own institution.
While the DRSC has thus far been successful in its mission, difficulties can arise when it comes to validating genes, Perrimon noted.
“In many cases, people come from the mammalian field … do a screen in Drosophila cells, and then [validate] the candidate genes in their mammalian system … using siRNAs or shRNAs,” he said. “But in other instances, people come from the Drosophila field and they want to go back in vivo and validate their candidate genes. This is where it could be a bit complicated.”
“In many cases, people come from the mammalian field … do a screen in Drosophila cells, and then [validate] the candidate genes in their mammalian system … using siRNAs or shRNAs. But in other instances, people come from the Drosophila field and they want to go back in vivo and validate their candidate genes. This is where it could be a bit complicated.”
For the 15,185 Drosophila genes, only about 20 percent have mutations, and even fewer have readily detectable phenotypes, Perrimon said. “So … if you go to the stock centers, you may not be able to find mutations in the gene that you are interested in.”
To help fill in this so-called phenotype gap, the DRSC launched the TRiP. According to the DRSC, the “lack of functional information on the majority of [Drosophila] genes does not indicate that these genes have no function. Instead, it indicates that we have been unable to either assay their roles experimentally or resolve an issue of functional redundancy.
“In addition, our understanding of a large fraction of ‘known’ genes is limited by pleiotropy, whereby the earlier function of a gene prevents its functional analysis at later developmental stages,” it said.
However, with transgenic RNAi, “it is now possible to disrupt the activity of single genes with a spatial and temporal resolution that is impossible or exceedingly difficult to achieve using classical genetic methods,” the DRSC added.
“What we’re doing is generating in vivo hairpin constructs … [and] setting up one RNAi line of flies for every gene in the [Drosophila] genome,” Perrimon said. “This is really the follow-up to the [DRSC so that] people now will be able to come up with a list of, let’s say, 100 candidate genes [and] we can give them 100 different fly lines that carry hairpin constructs against the genes they are interested in.”
He said the TRiP will take advantage of a system based on the phiC31 site-specific integration method for the conditional expression of RNAi hairpins in Drosophila that was developed by Perrimon and collaborators at Harvard.
“The hairpin constructs are being driven under the control of the UAS sequences, which allow people to express the hairpin only in tissues and cells that express Gal4,” Perrimon said. “We have thousands of lines that have been built by the community that express Gal4 in different cell types and tissues, so then, depending on what tissue people are interested in, they can do validation in that tissue or cell type.”
Although the TRiP was officially launched at the beginning of this month with the awarding of the NIH grant, Perrimon noted that creation of the lines started a few months with seed funding from the Howard Hughes Medical Institute, topping off a more than two-year effort to establish the methodology for the line-creation process.
“We spent quite a bit of time making sure everything was optimized in terms of level of expression of the transgene and so on,” he noted. “For the past four months, we’ve been starting the production of the lines [and] already have about 1,500 or 2,000 [lines] almost done.”
With the NIH grant, the TRiP will be able to bring that number to 6,250, and “hopefully, if we’re able to generate a little more money, we might be able to do it for every [Drosophila] gene,” Perrimon said.
“This is a good investment for the NIH,” he added. “It’s going to benefit the community.”