Identification of an RNA-dependent RNA polymerase in Drosophila establishes a common theme in RNA silencing.

In lower eukaryotes, such as A. thaliana, C. elegans, S.

Identification of an RNA-dependent RNA polymerase in Drosophila involved in RNAi and transposon suppression.

Here, we show that recombinant Drosophila elp1 (D-elp1) produced in Sf9 cells or Escherichia coli, corresponding to the largest of the three subunits in the RNA polymerase II core elongator complex, has RNA-dependent RNA polymerase (RdRP) activity. D-elp1 is a noncanonical RdRP that can synthesize dsRNA from different ssRNA templates using either a primer-dependent or primer-independent initiation mechanism. Of the three core subunits, only D-elp1 depletion inhibits RNAi in S2 cells but does not affect micro RNA function.

Injection of dsRNA into Drosophila Embryos for RNA Interference (RNAi).

INTRODUCTIONRNA interference (RNAi) is a powerful method for determining the role of specific genes during Drosophila embryogenesis. This protocol describes a technique by which Drosophila embryos can be injected with dsRNA in order to disrupt targeted gene function. The approach is straightforward, utilizing improved methods for injecting the dsRNA directly through the chorion of the embryo.

Collection of Drosophila Embryos for RNA Interference (RNAi).

INTRODUCTIONRNA interference (RNAi) is a powerful method for determining the role of specific genes during Drosophila embryogenesis. This protocol describes a method for collection of Drosophila embryos for RNA interference (RNAi) experiments. The embryos are collected in a simple, homemade apparatus, arrayed on prepared glass slides, and readied for injection.

Preparation of Double-Stranded RNA for Drosophila RNA Interference (RNAi).

INTRODUCTIONRNA interference (RNAi) is a powerful method for determining the role of specific genes during Drosophila embryogenesis. It has been used in our laboratory to phenocopy a series of known mutations in Drosophila, including twist, engrailed, daughterless, Dmef2, and, to a lesser extent, white in the adult eye. This protocol describes the preparation of dsRNA by in vitro transcription of complementary strands of a cloned DNA fragment that codes for all or a portion of the gene of interest, followed by annealing of the transcribed RNA.

Drosophila RNA Interference (RNAi) Using a Gal-4 Inducible Transgene Vector.

INTRODUCTIONRNA interference (RNAi) is a powerful method for determining the role of specific genes during Drosophila embryogenesis. This protocol describes a method for RNAi in vivo using tissue-specific Gal-4 transgenes to induce dsRNA synthesis from an upstream activator sequence (UAS) vector. This vector contains the desired exonic inverted sequences representing the target gene (preferably more than 400 bp) separated by a unique spacer, the first intron of the actin 5C gene.

Stereotypic founder cell patterning and embryonic muscle formation in Drosophila require nautilus (MyoD) gene function.

nautilus is the only MyoD-related gene in Drosophila. Nautilus expression begins around stage 9 at full germ-band extension in a subset of mesodermal cells organized in a stereotypic pattern in each hemisegment. The muscle founder cell marker Duf-LacZ, produced by the enhancer trap line rP298LacZ, is coexpressed in numerous Nautilus-positive cells when founders first appear.

Analysis of short interfering RNA function in RNA interference by using Drosophila embryo extracts and schneider cells.

The realization that short double-stranded RNA (dsRNAs) 21-25 bp in length represent the basis for posttranscriptional gene silencing (PTGS) in plants, quelling in N. crassa, and RNA interference (RNAi) in C. elegans and Drosophila has given insight into one of the most evolutionarily conserved pathways in eukaryotes.

The Drosophila selenoprotein BthD is required for survival and has a role in salivary gland development.

Selenium is implicated in many diseases, including cancer, but its function at the molecular level is poorly understood. BthD is one of three selenoproteins recently identified in Drosophila. To elucidate the function of BthD and the role of selenoproteins in cellular metabolism and health, we analyzed the developmental expression profile of this protein and used inducible RNA interference (RNAi) to ablate function.

Analysis of the 3(')-hydroxyl group in Drosophila siRNA function.

Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs.