JoVE Encyclopedia of Experiments
Microbiology
0 views • 2:44 min • August 31st, 2026
Begin with a mixture of plasmids suspended in a nutrient medium.
The first plasmid carries a recombinant adeno-associated virus (AAV) genome that encodes the gene of interest.
The second plasmid encodes the Rep and Cap proteins.
The third plasmid encodes helper proteins.
Add a transfection agent to the plasmid solution. Mix well and incubate.
The positively charged transfection agent binds to the negatively charged plasmid DNA, forming complexes.
Introduce the complexes to a monolayer of mammalian cells. Incubate.
The complexes enter the cells through endocytosis.
Within the cells, the plasmid-encoded genes drive the expression of viral proteins.
The Rep protein replicates the viral genome, while the Cap proteins form the capsid shells.
Rep protein, along with helper proteins, packages the replicated viral genome into capsids, forming complete AAV particles.
The recombinant AAV vectors carrying the gene of interest are ready for gene delivery applications.
After generating rAAV9 constructs and culturing HEK293 cells according to the text protocol, to transfect the cells on day one, combine 70 micrograms of AAV Rep-Cap plasmid, 70 micrograms of rAAV9 plasmid, and 200 micrograms of Ad helper plasmid in a 50-milliliter centrifuge tube. Add 49 milliliters of room temperature DMEM without FBS to the tube and mix well. Then, to make a four-to-one PEI to DNA solution, add 1, 360 microliters of PEI to the tube and mix well.
Incubate the solution at room temperature for 15 to 30 minutes. After the incubation, add five milliliters of the solution to each 150-milliliter dish of cells. Culture the cells at 37 degrees Celsius and 5%CO2 for 50 to 72 hours.