An In Vitro Transduction-Induced Myelination Assay for Oligodendrocyte Precursor Cells
Transkript
Take adherent cultures of primary oligodendrocyte precursor cells, or OPCs, in a growth medium.
Add control lentiviral vectors to the control well and test vectors, encoding signaling proteins that regulate myelination, to the test well, then incubate.
The cells internalize the vectors, and the viral RNA is reverse-transcribed into DNA and incorporated into the host genome, expressing the signaling proteins in the test cells.
Introduce enzymes to detach the OPCs from the substrate.
Neutralize the enzymes and centrifuge to remove the enzymes.
Add the growth medium and transfer the OPCs onto coverslips inside a microplate with adhered dorsal root ganglion or DRG neurons. Allow the OPCs to settle.
Fill the wells with the growth medium and incubate. Interaction with the DRG neurons triggers OPC maturation into oligodendrocytes.
The expressed signaling proteins induce oligodendrocytes to form myelin sheaths around the DRG neurons.
Assess the signaling protein-induced myelin sheath formation in the test cells compared to the control vector-transduced cells.
