Generation of Recombinant Adeno-Associated Virus Through Plasmid Transfection

Abstract

Source: Ding, J., et al. Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J. Vis. Exp. (2016)

This video demonstrates the production of recombinant adeno-associated virus particles through plasmid transfection in mammalian cells. It highlights the coordinated expression of Rep, Cap, and helper proteins that drive viral genome replication and capsid assembly within the nucleus.

Protocol

1. Transfection of HEK293 Cells with recombinant adeno-associated virus 9 (rAAV9) Plasmids

1. Prepare 1 µg/µl of linear polyethylenimine (PEI) solution. Dissolve PEI powder in endotoxin-free dH₂O that has been heated to 70-80 °C. After cooling down to RT, neutralize the solution to pH 7.0 with 1 M HCl. Filter sterilize (0.22 µm) the solution. Aliquot the 1 µg/µl PEI stock solution (1,400 µl/tube) and store the solution at -20 °C.
2. Culture HEK293 cells in Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. Culture the cells in a 37 °C incubator with 5 ± 0.5% carbon dioxide (CO₂).
3. At day 0, plate HEK293 cells in ten 150-mm dishes 18-20 hr before transfection by splitting > 90% confluent cells in a 1:2 dilution.
   NOTE: At day 1, the cells should reach 90% confluence.
4. At day 1, transfect HEK293 cells with the rAAV9 plasmid (e.g., rAAV9.cTNT::GFP or rAAV6.U6::shRNA constructs), Ad-Helper plasmid, and AAV-Rep/Cap plasmid using PEI.
   1. For 10 dishes of cells at 90% confluence, mix 70 µg of AAV-Rep/Cap plasmid, 70 µg of rAAV9 plasmid, and 200 µg of Ad-Helper plasmid in a 50-ml centrifuge tube.
   2. If the cells are less confluent, adjust the DNA amount proportionally. For instance, if the cells are at 75% confluent, reduce the DNA amount proportionally (75/90 of the amount shown in step 1.4.1): mix 70 x 75/90 = 58.3 µg of AAV-Rep/Cap plasmid, 70 x 75/90 = 58.3 µg of rAAV9 plasmid, and 200 x 75/90 = 166.7 µg of Ad-Helper plasmid in a 50-ml centrifuge tube.
   3. Add 49 ml of RT DMEM (without FBS) to the 50-ml tube and mix well.
   4. Add 1,360 µl of PEI solution to make the PEI:DNA ratio (v/w) be 4:1. Mix well. Incubate at RT for 15 - 30 min.
   5. Add 5 ml of the mixture prepared in step 1.4.4 to each 150-mm dish (50 ml of the mixture for ten 150-mm dishes).
5. Culture the cells in a 37 °C incubator with 5 ±0.5% CO₂ for 60-72 hr.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polyethylenimine, Linear (MW 25,000)	Polysciences, Inc.	#23966-2
Tube, Polypropylene, 36.2 ml, 25 x 87 mm, (qty. 56)	Beckman Coulter, Inc	# 362183
DMEM medium	Fisher Scientific	SH30243FS
Fetal Bovine Serum	Atlanta Biologicals	S11150
rAAV9 vector	Penn Vector Core	P1967