Biolistics for high-throughput transformation and RNA interference inDrosophila melanogaster

Abstract

With twelve Drosophila genomes now sequenced, there is a growing need to develop higher-throughput methods for identifying the functions of the many newly identified genes. Genetic transformation and RNA interference are two technologies that have been used extensively to facilitate gene-function studies in Drosophila melanogaster, to introduce genes or block the expression of endogenous genes, respectively. Both of these technologies typically require the delivery of nucleic acids into developing insect embryos, and virtually all studies to date have relied on microinjection as the DNA delivery method of choice. In this study, we describe the use of biolistics as a higher-throughput method of nucleic acid delivery. By bombarding dechorionated D. melanogaster embryos with 1 μm gold beads coated with P-element or piggyBac transformation vectors, we observed transformation frequencies (3-4%) that are comparable to those achieved using microinjection methods, but in only a fraction of the time required for the DNA delivery. Biolistic delivery of double-stranded RNA (dsRNA) specific to a β-glucuronidase (gus) transgene resulted in a significant (71%) reduction in gus transcripts in embryos and the RNA interference (RNAi) persisted through two successive larval molts, albeit at reduced levels. DsRNAs specific to four essential genes were delivered to embryos and resulted in arrested development and phenotypes that closely match that of null mutations. These results suggest that biolistic delivery of dsRNA into embryos could be adapted for high throughput RNAi screens of early Drosophila developmental genes.

Ingested double-stranded RNAs can act as species-specific insecticides

“…Previously, we have shown that injection or biolistic delivery of dsRNAs that target various genes essential to cell viability resulted in death of developing D. melanogaster embryos and larvae (Yuen et al, 2008). Ingested dsRNA has previously been shown to be lethal or produce deleterious effects in a limited number of insect species thus far (Baum et al, 2007;Mao et al, 2007;Zhou et al, 2008;Walshe et al, 2009), but not in drosophilids.…”

“…A transgenic strain of D. melanogaster carrying a ubiquitouslyexpressed b-glucuronidase gene (gus) under the control of the act5C promoter (Yuen et al, 2008) was used to test the efficacy of several different methods of dsRNA delivery, including feeding. By targeting a transgene that has no observable effect on the viability or fitness of the insects, RNAi could be induced without adversely affecting their growth or development.…”

“…A gus transgenic strain of D. melanogaster (Yuen et al, 2008), carrying the Escherichia coli b-glucuronidase (gus) gene under the control of the D. melanogaster actin 5c promoter was the host used for the delivery of gus-dsRNA. All cultures were reared on an agar-yeast-cornmeal medium at room temperature on a 12 h light: 12 h dark photoperiod.…”

“…Eaton et al (2002) observed gene silencing in Drosophila dechorionated embryos soaked in dsRNA, although no other researchers have since used this technique to deliver dsRNA. Biolistics has also been shown to be effective at delivering dsRNA into Drosophila embryos (Yuen et al, 2008), but this technique has not yet been used for any other species. Oral delivery of dsRNA was first demonstrated in C. elegans (Timmons et al, 2001), and has since been adapted for a limited number of insect species.…”

Biolistic Transformation for Delivering DNA into the Mitochondria

“…The technique has become a widely utilized system for the transformation of a wide range of organisms including insects (Yuen et al 2008 ), plants (Becker et al 2000 ;Fernando et al 2000 ;Fukuoka et al 1998 ;Klein et al 1988 ;Maenpaa et al 1999 ;RascoGaunt et al 1999 ;Tang et al 1999 ), animals such as Caenorhabditis elegans (Isik and Berezikov 2013 ), animal cells (Johnston 1990 ), algae (Mayfi eld and Kindle 1990 ), bacteria (Shark et al 1991 ), fungi (Bills et al 1995 ;Durand et al 1997 ;Fungaro et al 1995 ;St. Leger et al 1995 ), and intracellular organelles Larosa and Remacle 2013 ).…”