This article details a method for transducing mature human neurons with lentiviral vectors encoding a mutant tau protein fused to a yellow fluorescent protein (YFP). The process enables the study of tau protein aggregation in neuronal cells over time.
Begin with an adherent culture of mature human neurons in a matrix-coated plate.
Add lentiviral vectors encoding a mutant human tau protein fused to a yellow fluorescent protein (YFP) reporter.
The lentivirus attaches to specific neuronal cell surface receptors, enabling viral-host membrane fusion and the release of viral RNA and enzymes.
The viral RNA is reverse-transcribed into DNA.
Viral integrase transports the DNA into the nucleus, facilitating integration into the host genome, and leading to the production of YFP-tagged mutant tau protein.
Over time, the mutant tau forms cytoplasmic aggregates in the transduced cells.
Wash the cells with fresh media to remove non-internalized viral particles.
Continue incubation with regular media changes to maintain neuronal viability and promote protein aggregation.
Visualize the neurons under a microscope.
Successfully transduced neurons exhibit cytoplasmic aggregates of fluorescent YFP-tagged mutant tau protein.
To transduce neurons with lentivirus, use a titer count of 340,000 transduceable units per cell. Dilute the transduceable units in cell culture media to the necessary concentration, and add them to the cells.
Two days after adding the lentivirus, wash the cells once with fresh media that does not contain bFGF.
Continue culturing the cells at 37 degrees Celsius with 5% carbon dioxide in media without bFGF. Maintain the cells for about eight weeks after transduction, making sure to change the cell culture media every other day. Use a light microscope to routinely visualize the cells and ensure viability.
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