JoVE Encyclopedia of Experiments
Microbiology
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Take early-instar mosquito larvae, which are susceptible to viral infection.
Transfer the larvae to a beaker containing deionized water.
Introduce recombinant densovirus, engineered to encode a microRNA that suppresses target larval genes. Incubate.
The larvae take up the virus, which then reaches the gut.
The ingested virus crosses the gut epithelium and spreads to other tissues.
The virus enters larval cells via endocytosis and releases its DNA into the nucleus.
The viral DNA produces microRNAs.
In the cytoplasm, these microRNAs bind to the RNA-induced silencing complex.
The complex binds and cleaves complementary target larval mRNAs, preventing expression and leading to gene silencing.
After incubation, wash the larvae with deionized water to remove non-ingested viral particles.
Transfer the larvae to a fresh plate with food for recovery.
The virus-infected mosquito larvae, with the target gene silenced, are ready for downstream applications.
Wash 100 first-instar A. albopictus larvae three times, using deionized water. And then, transfer the larvae into a beaker containing 95 milliliters of deionized water.
Add five milliliters of the rAaeDV stocks, and incubate the larvae for 24 hours at 28 degrees Celsius. For success of larva infection, it is essential for larvae to be contained with infection virus particles for 24 to 36 hours to ensure high infection ratios. Following the incubation, wash the larvae three times using deionized water, and then transfer the larvae back to the plates. Feed the larvae regularly.