Harvesting Recombinant Aedes aegypti Densovirus from Infected Cells

Résumé

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 process of isolating recombinant Aedes aegypti densovirus (rAaeDV) from infected mosquito-derived epithelial cells. The infected cells are collected and exposed to repeated freeze-thaw cycles for lysis. Lysate is centrifuged, and the supernatant containing viral particles is filtered, aliquoted, and stored at an ultra-low temperature for future use.

Protocole

1. Transfecting C6/36 Cells with Aedes aegypti densovirus (rAaeDV) Plasmids

1. Culture C6/36 cells in Roswell park memorial institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 28 °C incubator.
2. On day 0, plate the cells in ten 25 cm² culture flasks 18-20 h before transfection by splitting 90-95% confluent cells in a 1:2 dilution.
   NOTE: By day 1, the cells should reach 90-95% confluence.
3. On day 1, transfect the cells with the series of rAaeDV plasmids.
   1. Dilute 10 µg DNA in 600 µL of RPMI-1640 medium in a microcentrifuge tube and mix gently. Meanwhile, prepare 600 µL of RPMI-1640 medium and 25 µL of transfection reagent per transfection.
   2. Incubate for 5-10 min at room temperature (RT). Then, add 625 µL of the 1640- transfection reagent mixture to the RPMI-1640-plasmid DNA mixture.
   3. Incubate this mixture for 20-30 min at RT. Before transfection, wash the cells twice with 2 mL of RPMI-1640 medium.
   4. After the RT incubation (step 1.3.3), add the RPMI-1640-transfection reagent DNA plasmid mixture to the 25 cm² culture flask and incubate for 6 h at 28 °C.
   5. At approximately 6 h post-transfection, remove the transfection media, wash the cells twice with 5 mL of RPMI-1640 medium, and then add RPMI-1640 medium with 10% FBS.
      NOTE: rAaeDV shows high rates of infection (95%) in Ae. albopictus larvae; however, in our experience, in nearly 50% of infected larvae, the infection was restricted to primary infection sites (e.g., anal papillae and bristle cells) without dissemination. Therefore, if there is a need to infect larvae in multiple tissues, a defective recombinant virus expressing fluorescent protein should be co-infected to enable the visualization of infection sites. For example, to produce a defective recombinant virus expressing DsRed, p7NS1-DsRed should be co-transfected with a helper plasmid into C6/36 cells.

2. Harvesting rAaeDV Virions from Transfected C6/36 Cells

1. Harvest the cells 5 days after transfection. Dislodge and suspend the cells in the dishes using a 5 mL glass dropper, pipetting up and down with the culture medium. Transfer all cell suspensions to sterile 15-mL tubes.
2. Freeze at -80 °C or in a dry ice/ethanol bath for 30 min and then thaw at 37 °C. Vortex for 1 min. Repeat the freeze-and-thaw procedure 3 times.
3. Centrifuge the sample at 4,800 x g for 20 min at 4 °C. Collect the supernatant and discard the pellet. Then, pass the supernatant through a 0.22 µm disposable syringe filter. Aliquot and store the final purified virion stocks at -80 °C.
   NOTE: Avoid repeated freeze-thaw cycles.

Matériaux

Liste des matériaux utilisés dans cet article

Nom	Entreprise	Numéro de catalogue	Commentaires
Centrifuge machine	Thermo Scientific	75004260
Centrifuge System	Beckman Coulter	363118