Infecting Primary Mouse Midbrain Neurons with Engineered Adeno-Associated Viruses
Transkript
Begin with an embryonic mouse midbrain cell culture on a coverslip.
Treat with engineered adeno-associated viruses encoding a fluorescent calcium indicator complex under a neuron-specific promoter.
Incubate for viral attachment and internalization.
Remove the medium to eliminate unbound viruses, then add a culture medium and incubate.
Internalized viruses release their genome in the nucleus, where the neuron-specific promoter drives the production of calcium indicator complexes exclusively in primary neurons.
These complexes include a fluorescent protein tagged with a calcium-binding protein and an M13 peptide.
Transfer the coverslip to a dish with a recording buffer rich in calcium ions, then place it into a recording chamber for live confocal imaging.
Introduce a glutamate recording buffer, causing glutamate to bind with ion channels, leading to the entry of calcium ions.
These ions bind to the complex, causing a conformational change and fluorescence emission.
In live imaging, the bright neurons indicate the viral infection.
