Transfection of Wolbachia pipientis into Drosophila Embryos

Stephen L. Dobson1

1 University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 3A.4
DOI:  10.1002/9780471729259.mc03a04s05
Online Posting Date:  June, 2007
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Abstract

Wolbachia is a genus of obligate intracellular Alpha‐Proteobacteria represented by the type species Wolbachiapipientis (Dumler et al., 2001). Wolbachia commonly reside within cytoplasmic vacuoles of arthropods and helminths (Werren and Windsor, 2000; Casiraghi et al., 2004); vertebrate infections have not been identified. Wolbachia are maternally transmitted from mothers to offspring though the embryonic cytoplasm. Wolbachia are able to induce a diverse range of phenotypes in their invertebrate hosts, ranging from classical mutualism to reproductive parasitism. Examples of the latter include male killing, host feminization, parthenogenesis, and cytoplasmic incompatibility (reviewed in Dobson, 2003a). Current Wolbachia research foci include examining the impacts of Wolbachia infection on host evolution, characterizing the mechanisms by which Wolbachia manipulate invertebrate hosts, and developing applied strategies that employ Wolbachia for pest and disease control. Wolbachia transfection has proven a useful technique for addressing questions within each of these research foci. This unit describes a method for Wolbachia transfection via embryonic microinjection.

Keywords: cytoplasmic incompatibility; population replacement; filariasis

     
 
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Table of Contents

  • Basic Protocol 1: Microinjection of Drosophila Embryos
  • Support Protocol 1: Collection of Drosophila Embryos
  • Support Protocol 2: Preparation of Apple Juice Plates
  • Support Protocol 3: Preparation of Microinjection Needles
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Microinjection of Drosophila Embryos

  Materials
  • Wolbachia‐infected and ‐uninfected Drosophila embryos ( protocol 2)
  • Apple juice agar plates ( protocol 3; Fig. )
  • 50% bleach solution (Clorox bleach recommended)
  • Halocarbon oil 700 (Sigma)
  • Uninfected Drosophila males
  • Camel hair paint brush (Fig. ) or alternate preferred tool for manipulating embryos
  • Fine mesh for embryo collection and washing
  • Filter paper, damp
  • Double‐sided tape (e.g., Scotch, #666)
  • Glass slides
  • Microinjector, microinjection needles ( protocol 4; Fig. D,E) and micromanipulator
  • Dissecting microscope
  • Compound microscope with movable stage

Support Protocol 1: Collection of Drosophila Embryos

  Materials
  • Agar
  • Sucrose
  • Apple juice
  • 10% Tegosept (optional, Fisher Scientific)
  • Live baker's yeast
  • 35 × 10–mm petri plates (BD Falcon)

Support Protocol 2: Preparation of Apple Juice Plates

  Materials
  • Micropipet puller (Sutter Instrument Co., P‐87)
  • Borosilicate glass capillaries (World Precision Instruments, TW100F‐4)
  • Micropipet beveller (Sutter Instrument Co., BV‐10)
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Figures

Videos

Literature Cited

Literature Cited
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