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 pla
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