Silencing of genes in cultured Drosophila neurons by RNA interference

Abstract

Cultures of neuroblasts that generate abundant neurons were established from Drosophila embryos to study silencing of genes by RNA interference (RNAi). Cultured cells expressed ELAV, a marker of neurons, Futsch, a marker of neurites, and Synapsin, Synaptobrevin, and Synaptogamin, proteins involved in neurotransmitter secretion. Conditions were found for efficient transfection of cells with siRNAs for ELAV or the insulin-like receptor, which resulted in marked decreases in neurons that express ELAV and Futsch. Cells also were successfully transfected with long-chain Sox-Neuro dsRNA resulting in a 55% reduction of neurons expressing Futsch. The results suggest that this cultured neural cell system can be used to study RNAi-dependent silencing of genes involved in many kinds of neural functions.ELAV ͉ insulin receptor ͉ neurites G enome-wide RNAi screens using clonal lines of cultured Drosophila cells have been used to identify genes involved in cell growth and viability (1), signaling pathways (2-12), and factors required for bacterial infection (13). RNAi screens in intact Drosophila embryos also have been used to identify genes that play roles in neural development (14-15) and cardiac development (16).We have been using RNAi to find genes that are involved in the assembly of the Drosophila embryonic nervous system. It is much easier and faster to screen tissue culture cells by RNAi by transfecting cells with double-stranded oligoribonucleotides (siRNA) or long-chain dsRNA than to laboriously inject embryos with RNA preparations. However, well differentiated, well characterized clonal lines of Drosophila neural cells are needed.As an alternative, we have dissociated neuroblasts (NBs) from Drosophila embryos and cultured them under conditions that result in an enriched population of NBs that generate abundant neurons in vitro and that can be transfected with siRNA or long-chain dsRNA with relatively high efficiency. Cultures enriched in NBs and neurons provide a means of studying molecular and cellular events during nervous system development (17). NB cultures have been used successfully to study gene expression during NB lineage development (18) and to explore mechanisms controlling asymmetric cell division and protein localization (19). Cultured Drosophila motor neurons also have been shown to form synapses with striated muscle cells (20). Specific cells have been removed from embryos with capillaries and have been grown in culture to study their development state (21), ion channels and responses to neurotransmitters, neuronal morphology, and action potential-dependent synaptic activity (22). K. Sepp also has screened cultured fluorescent Drosophila embryo neurons by RNAi (http://flyrnai.org).Our main objective in this study has been to devise conditions that enable mass cultures of Drosophila neural cells to be used for RNAi-induced silencing of genes. Results and DiscussionDrosophila embryos Ϸ5 h after fertilization (stage 10) were used to establish highly enriched NB cultures by a slight modification of publi...

RNA interference screening inDrosophilaprimary cells for genes involved in muscle assembly and maintenance

“…Importantly, RNAi screens in Drosophila primary cultures can be carried out by a simple bathing method for dsRNA uptake. This is in contrast to the difficulties that have been reported with RNAi in mammalian cell lines and primary cells, which requires delivery of siRNAs into cells by chemical transfection or electroporation (Ovcharenko et al, 2005;Sharma and Nirenberg, 2007).…”

“…As the different cell types can be tracked in primary cultures using a tissuespecific GFP, antibodies or other markers, primary-cell-based RNAi screens may be used to identify genes required in other differentiated cells as well [e.g. primary neurons (K. Sepp and N.P., unpublished) (Sharma and Nirenberg, 2007)]. Importantly, RNAi screens in Drosophila primary cultures can be carried out by a simple bathing method for dsRNA uptake.…”

Evidence That Myosin Activity Opposes Microtubule-Based Axonal Transport of Mitochondria

“…RNA interference (RNAi) using double-stranded RNA (dsRNA) is a remarkably effective tool for suppressing specific gene expression in Drosophila cells (Goshima and Vale, 2003; Rogers et al, 2003; Sharma and Nirenberg, 2007). However neurons are generally thought to be less amenable to RNAi than other cell types.…”

Codes and Circuits

“…His contributions to neuroscience were profound, and included the development and utilization of cell lines in neuroscience (Nelson et al 1976(Nelson et al , 1969Nirenberg 1978;Nirenberg et al 1983); the neuroblastoma and hybrid lines and the PC-12 cells developed by Lloyd Greene in Marshall's lab have been used in thousands of studies. He and his colleagues provided an useful cellular model of opiate addiction (Sharma et al 1975(Sharma et al , 1977 and have identified a number of novel genes that are important for nervous system development (Koizumi et al 2007;Sharma and Nirenberg 2007). They have identified new homeodomain genes (Kim and Nirenberg 1989;Rovescalli et al 1996).…”