Recombinant Densovirus-Mediated Gene Silencing in Mosquito Larvae

Abstract

Source: Liu, P., et al. Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae. J. Vis. Exp. (2017)

This video demonstrates the use of recombinant mosquito densoviruses encoding microRNA to silence specific genes in mosquito larvae. Infected early instar larvae produce functional microRNAs that suppress target gene expression for downstream studies.

Protocol

1. Mosquito Transduction

1. Wash 100 1st instar Aedes albopictus larvae three times using deionized water, and then, transfer the larvae into a beaker containing 95 mL deionized water. Add 5 mL of the recombinant Aedes aegypti densoviru (rAaeDV) stocks (final concentration of 1.00 x 10¹⁰ copies/mL).
2. Incubate for 24 h at 28 °C, wash the larvae three times using deionized water, and then transfer the larvae back to the plates and feed regularly.
3. Monitor the level of target gene expression in the larvae with qPCR or Western blot.
   NOTE: The fluorescence signal detection method can be used to isolate the infected larvae more precisely. For example, to detect fluorescent signals from larvae infected with recombinant virus expressing DsRed, the larvae can be placed on a concavity slide and observed under an inverted fluorescence microscope every 8 h until 2 days post-infection. Once fluorescent larvae are detected, they should be separated into individual plastic test cups to allow for subsequent continuous observation. Larvae with multiple tissues infected can be identified using this method.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Centrifuge machine	Thermo Scientific	75004260
Centrifuge System	Beckman Coulter	363118
SuperReal PreMix Plus (SYBR Green)	Beijing Tiangen Biotech	FP205