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

Samenvatting

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

1. Infect flies by nano-injection and perform survival assays.
   1. Pull the capillaries to prepare injection needles, back-fill the injection needle with oil, and assemble the injector.
   2. 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 vial).
      NOTE: The injection is time-consuming (generally 100 flies per hour) and Drosophila C virus (DCV) replicatio....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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