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RNA interference (RNAi) is the phenomenon of reduction in expression (knockdown) by double stranded RNA (dsRNA) of genes with sequence homology to the dsRNA. Andrew Fire and Craig C. Mello were awarded the Nobel Prize in Physiology or Medicine in 2006 for their discovery of RNAi in the nematode C. elegans. Although the physiological role of RNAi as well as its molecular mechanism are highly interesting, our lab’s main interest in RNAi lies in its use as a research tool to knockdown gene expression in tissue culture cells (or organisms), making it possible to study the contribution of individual genes to cellular processes with relative ease.
Studying the effects of RNAi-mediated gene knockdowns in Drosophila tissue culture cells offers several advantages over mammalian cells. Drosophila lacks the interferon-response that causes many mammalian cells to shutdown transcription when challenged with long (> 21bp) dsRNAs. Therefore, 400-1000bp dsRNAs can be used to elicit the RNAi response in Drosophila cells. These long dsRNAs can be synthesized quickly and affordably by a single PCR reaction using genomic DNA as a template, followed by in vitro transcription. Also, several Drosophila cell lines take up these long dsRNAs directly from the culture medium, making the procedure to elicit the RNAi response very simple and amenable to high-throughput techniques. Since Drosophila cells grow at room temperature, observation of living cells through the microscope is straight-forward and does not need complicated temperature and atmospheric controls (as in the case of mammalian cells). Also, it is possible to let Drosophila cells adhere to glass slides, enabling high-resolution microscope observations of sub-cellular structures.
In 2002 we developed, together with the labs of Pat O'Farrell and Graeme Davis a library of dsRNA molecules targeting ~ 7000 Drosophila genes that have homology to either human or C. elegans genes. This library has been used in a number of screens at UCSF (see references, below) and is now referred to as UCSF v.1Drosophila RNAi library. It is distributed by Open Biosystems both in the form of DNA and RNA. A comprehensive description of the library can be found here.
In 2004 our lab discovered that many "hits" (in a screen for mitotic index changes) were caused by dsRNAs containing trinucleotide repeats (most often coding for poly-glutamate stretches). This observation made us realize that 1: these low complexity regions were accidentally represented in the RNAi library, and that 2: these regions obviously contributed to knock-down of targets other than the intended gene. We used this experience in the design of UCSF v.2 Drosophila which targets all genes of the Drosophila genome. This library was designed in January 2005 (by Nico Stuurman), and over 90% of the ~15000 dsRNAs in this library do not contain any 21bp sequence also found elsewhere in the genome. This library has been synthesized at Open Biosystems (with help by Gohta Goshima and Yi Guo), and is available both in the form of DNA and RNA.
updated 4/9/07
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