果蝇的大肠埃希杆菌感染

Charles  Tracy; Helmut  Krämer

doi:10.21769/BioProtoc.2256

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Peer-reviewed

Escherichia coli Infection of Drosophila

果蝇的大肠埃希杆菌感染

CT Charles Tracy

Helmut Krämer email

发布: 2017年05月05日第7卷第9期 DOI: 10.21769/BioProtoc.2256 浏览次数: 8407

评审: Ruth A. FranklinModesto Redrejo-RodriguezFilipa Vaz

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参见作者原研究论文

The authors used this protocol in:

Cover of Immunity, featuring study using the protocol.

Aug 2016

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Abstract

Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with important roles in innate immunity. The following protocol is designed to reproducibly inject a known amount of non-pathogenic E. coli into otherwise sterile flies and to measure the survival of flies after infection. The protocol can be easily adapted to different types of bacteria.

Keywords: Drosophila (果蝇)

Innate immunity (天然免疫)

Bacterial infections (细菌感染)

Tolerance (耐受性)

Survival (生存)

Background

Classic infection experiments involve infecting Drosophila orally (Chakrabarti et al., 2016) or with a needle dipped in a concentrated bacterial solution (Romeo and Lemaitre, 2008). Unlike these protocols, our experimental procedure allows us to determine the site of infection and precisely control the dose of bacteria injected into each fly. This provides homogeneity and reproducibility, and allows us to adapt bacterial load for different experiments (Akbar et al., 2011 and 2016).

Materials and Reagents

15 ml Falcon tubes (Corning, catalog number: 352196 )
1.5 ml Eppendorf tubes (USA Scientific, catalog number: 1615-5500 )
Kimwipes
26 G needle (BD, catalog number: 305111 )
Spin columns (e.g., Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 69700 )
Empty and clean Drosophila vials (Genesee Scientific, catalog number: 32-109 )
Microloader tips (Eppendorf, catalog number: 930001007 )
Vials (Genesee Scientific, catalog number: 32-109 ) with standard Drosophila food (lightly yeasted)
Microslide (Corning, catalog number: 2948-75X25 )
50 Drosophila melanogaster adult virgins (Romeo and Lemaitre, 2008), aged five to seven days post eclosion (see Note 1)
E. coli-DH5α containing any ampicillin-resistant plasmid (e.g., expressing GFP)

pEGFP (https://www.addgene.org/vector-database/2485/)
pET-GFP-C11 (http://www.addgene.org/30183/)

LB broth (Fisher Scientific, catalog number: BP1426-500 )
Ampicillin (200 mg/ml) (Sigma-Aldrich, catalog number: A9518 )
70% ethanol–diluted from 100% ethanol (Pharmco-AAPER, catalog number: 111000200 )
Mineral oil (Fisher Scientific, catalog number: O121-1 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O) (Sigma-Aldrich, catalog number: S9390 )
Potassium phosphate monobasic (KH₂PO₄) (Fisher Scientific, catalog number: S397-500 )
1x PBS (see Recipes)

Equipment

37 °C incubator with shaking (Eppendorf, New Brunswick^TM, model: Innova^® 44 )
Spectrophotometer (Molecular Devices, model: SpectraMax M2 )
Borosilicate glass capillaries (WPI, catalog number: TW100-4 )
Centrifuge capable of spinning Falcon tubes (Eppendorf, model: 5804 R )
Flaming/Brown Micropipette puller (Sutter Instrument, model: P-97 )
Mini benchtop centrifuge (Fisher Scientific, model: Fisherbrand^TM Standard Mini Centrifuge , catalog number: 05-090-100)
Pico-Injector (Nikon Instruments, catalog number: PLI-188 )–requires nitrogen gas
Note: Should have foot pedal for injecting.
25 °C incubator (BioCold Environment, model: BC49-IN )
Anesthetizing fly pad (Genesee Scientific, catalog number: 59-119 )
Small brush
Dissecting microscope (Leica)