Biolistics for high-throughput transformation and RNA interference in Drosophila melanogaster (original) (raw)
Related papers
Injection of dsRNA into Drosophila Embryos for RNA Interference (RNAi)
Cold Spring Harbor …, 2008
RNA 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 ...
Insects, 2013
RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in
Biochemistry and Molecular Biology Education, 2010
RNA mediated gene interference (RNAi) is now a key tool in eukaryotic cell and molecular biology research. This article describes a five session laboratory practical, spread over a seven day period, to introduce and illustrate the technique. During the exercise, students working in small groups purify PCR products that encode in vitro transcription promoters fused to sequences from Drosophila genes of the Rho/Rac GTPase family. These DNA templates are then used to synthesize double stranded RNAs (dsRNA), which are subsequently used to transfect Drosophila Kc embryonic cells. The resulting RNAi produces simple cellular phenotypes that are observed following fluorescent histochemical staining. These phenotypes are ultimately related to gene ontology data that the students generate through a bioinformatic analysis of the sequences transcribed into dsRNA. Taken together, this laboratory exercise provides ''hands on'' experience of RNAi in a class setting and provides a framework for the in-depth discussion of how this technique can be applied to studies of gene function.
Preparation of Double-Stranded RNA for Drosophila RNA Interference (RNAi)
Cold Spring Harbor Protocols, 2008
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.
Delivery of dsRNA for RNAi in insects: an overview and future directions
Insect Science, 2013
RNA interference (RNAi) refers to the process of exogenous double-stranded RNA (dsRNA) silencing the complementary endogenous messenger RNA. RNAi has been widely used in entomological research for functional genomics in a variety of insects and its potential for RNAi-based pest control has been increasingly emphasized mainly because of its high specificity. This review focuses on the approaches of introducing dsRNA into insect cells or insect bodies to induce effective RNAi. The three most common delivery methods, namely, microinjection, ingestion, and soaking, are illustrated in details and their advantages and limitations are summarized for purpose of feasible RNAi research. In this review, we also briefly introduce the two possible dsRNA uptake machineries, other dsRNA delivery methods and the history of RNAi in entomology. Factors that influence the specificity and efficiency of RNAi such as transfection reagents, selection of dsRNA region, length, and stability of dsRNA in RNAi research are discussed for further studies.
In vitro analysis of RNA interference in Drosophila melanogaster
Methods, 2003
Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21-to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.
Molecular Psychiatry, 2001
RNA interference (RNAi) is a gene silencing mechanism that can be triggered by introducing double-stranded RNA (dsRNA) into cells expressing the appropriate molecular machinery, which then degrades the corresponding endogenous mRNA. RNAi can be used for determining gene function and creating functional 'knockout' organisms. Here we show for the first time that RNAi can be induced in adult fruit flies by injecting dsRNA into the abdomen of anesthetized Drosophila, and that this method can also target genes expressed in the central nervous system (CNS). Two genes were targeted to investigate the effects of dsRNA injection on their mRNA content; lacZ transgene (expressed either in the gut or in the CNS), and GM06434, the Drosophila homologue of the C. elegans gene nrf (nose resistant to fluoxetine). Both the transgene and the endogenous gene were successfully silenced in adult Drosophila by intra-abdominal injection of their respective dsRNA. We propose that our method of RNAi in adult flies can be used to characterize gene functioning in the CNS without the typical interference in development found in most gene mutation studies. Molecular Psychiatry (2001) 6, 665-670.
Journal of Biological Chemistry, 2006
Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells. . 2 The abbreviations used are: dsRNA, double-stranded RNA; AcLDL, acetylated low density lipoprotein; Chc, clathrin heavy chain; CHO, Chinese hamster ovary; DEAD-box, Asp-Glu-Ala-Asp sequence; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; RISC, RNA-induced silencing complexes; RNAi, RNA interference; siRNA, small interfering RNA; PBS, phosphate-buffered saline; BSA, bovine serum albumin.