JoVE Encyclopedia of Experiments
Microbiology
0 views • 3:55 min • September 30th, 2026
Take a culture of human cancer cells expressing an endogenous target protein and remove the medium.
Take a suspension of recombinant adenovirus carrying a gene that encodes the fluorophore-tagged target protein.
Add a small volume of the viral suspension to the cells to ensure close contact between the virus and the cell surface.
Incubate with occasional shaking to allow viral attachment.
Add additional medium to the culture.
Next, introduce an siRNA–calcium phosphate complex to target the mRNA of the endogenous target protein and incubate.
The calcium phosphate enhances siRNA uptake, and the siRNA is loaded into RISC.
The siRNA–RISC complex then binds to and cleaves the target mRNA, inhibiting protein expression.
Simultaneously, the adenovirus enters the cell and releases its DNA, which directs the expression of the fluorophore-tagged target protein.
This enables endogenous target protein depletion and simultaneous replacement with the fluorophore-tagged protein.
Wash the cells with buffer and perform fluorescence imaging to analyze the target protein.
Label a 1.5 milliliter microcentrifuge tube for each condition, and add 400 microliters of warm alpha MEM minus. After slowly thawing aliquot
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