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…
Offenlegungen
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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Diesen Artikel zitieren
Intrathoracic Viral Nano-Injection to Study Host-Virus Interactions in Adult Flies. J. Vis. Exp. (Pending Publication), e21614, doi: (2023).