Home › Microbiology › RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells
Microbiology JoVE (Open Access) Citable · DOI

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

DOI: 10.3791/2137-v
What you'll learn
  • ✓ Design and execute genome-wide RNAi screens in Drosophila cells
  • ✓ Perform vaccinia virus infection assays in 384-well plate format
  • ✓ Identify and interpret host factors affecting viral infection rates
  • ✓ Apply loss-of-function screening to virology research
Protocol

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Difficulty
advanced
Total time
~5-7 days (RNAi knockdown kinetics + viral infection + staining + analysis)
Model organism
Drosophila melanogaster (S2 cells)
Biosafety
BSL-2

Steps

1
Introduce RNA interference screening principles

Overview of cell-based genome-wide RNAi screening methodology and advantages of Drosophila system for identifying host factors in viral infection.

▶ 00:58
2
Execute RNAi knockdown in 384-well plates

Transfect dsRNA targeting specific genes into Drosophila cells in high-throughput 384-well plate format to achieve efficient knockdown of host factors.

▶ 01:19
3
Infect cells with vaccinia virus

Apply vaccinia virus to RNAi-treated Drosophila cells at appropriate multiplicity of infection and incubate to allow viral replication.

▶ 03:09
4
Stain and fix infected cell cultures

Fix infected cells and perform immunostaining to visualize viral antigens or cellular markers for quantifying infection rates across all wells.

▶ 04:11
5
Analyze infection rates and identify hits

Image stained plates and interpret representative results to identify RNAi targets that significantly alter vaccinia virus infection susceptibility.

▶ 06:02
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