Biolistics for high-throughput transformation and RNA interference in Drosophila melanogaster.
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 microm 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 beta-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.
Yuen
Jenna L
JL
Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba, Canada.
Read
Scott A
SA
Brubacher
John L
JL
Singh
Aditi D
AD
Whyard
Steven
S
D000818:Animals; D019470:Biolistics / Q000379:methods; D004247:DNA / Q000235:genetics; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics; D008845:Microinjections / Q000379:methods; D034622:RNA Interference / Q000502:physiology; D012330:RNA, Double-Stranded / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes
D017931:DNA Primers; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D004247:DNA; D005966:Glucuronidase