Intrathoracic Viral Nano-Injection to Study Host-Virus Interactions in Adult Flies

Published: August 31, 2023

Abstract

Source: Yang, S., et al. Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster. J. Vis. Exp. (2019).

In this video, we describe a procedure to perform a nano-injection of a positive-sense, single-stranded RNA virus into adult male Drosophila flies and subsequently study the antiviral responses initiated due to the infection. Polymerase chain reaction analysis identifies the upregulation of specific genes in the infected flies that indicate the activation of immune response pathways including RNA interference and JAK/STAT signaling.

Protocol

1. Viral Infection in Drosophila Infect flies by nano-injection and perform survival assays. Pull the capillaries to prepare injection needles, back-fill the injection needle with oil, and assemble the injector. Anaesthetize flies on the pad under a light flow of CO2. Inoculate flies by intra-thoracic injection of 50.6 nL of virus solution (100 PFU, diluted with Tris-HCl buffer, pH 7.2). Inject at least 3 vials of flies (20 flies per…

Divulgations

The authors have nothing to disclose.

Materials

Microscope Olympus CKX41
Nanoject II Auto-Nanoliter Injector Drummond Scientific 3-000-204 Nanoject II Variable Volume (2.3 to 69 nL) Automatic Injector with Glass Capillaries (110V)
Schneider's Insect Medium Sigma S9895 Cell culture
10 cm cell culture dish Sigma CLS430167 Cell culture
Drosophila Incubator Percival I-41NL Rearing Drosophila
0.22um filter Millipore SLGP033RS
1.5 ml Microcentrifuge tubes Brand 352070
1.5 ml RNase-free Microcentrifuge tubes Axygen MCT-150-C
ABI 7500 qPCR system ABI 7500 qPCR
Centrifuge Eppendorf 5810R
Centrifuge Eppendorf 5424R
Chloroform Sigma 151858 RNA extraction
DEPC water Sigma 95284-100ML RNA extraction
Isopropyl alcohol Sigma I9516 RNA extraction
Lysis buffer (RNA extraction) Thermo Fisher 15596026 TRIzol Reagent
Lysis buffer (liquid sample RNA extraction) Thermo Fisher 10296028 TRIzol LS Reagent
Optical Adhesive Film ABI 4360954 qPCR
Penicillin-Streptomycin, Liquid Invitrogen 15140-122 Cell culture
qPCR plate ABI A32811 qPCR
Statistical software GraphPad Prism 7
TransScript Fly First-Strand cDNA Synthesis SuperMix TransScript AT301 RNA extraction
Vortex IKA VORTEX 3 RNA extraction
FBS Invitrogen 12657-029 Cell culture
Flat bottom 96-well-plate Sigma  CLS3922  Cell culture
Cell Incubator Sanyo MIR-553
100 Replacement tubes Drummond Scientific 3-000-203-G/X

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Citer Cet Article
Intrathoracic Viral Nano-Injection to Study Host-Virus Interactions in Adult Flies. J. Vis. Exp. (Pending Publication), e21614, doi: (2023).

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