Lentiviral Transduction of Neural Progenitor Cells for Epigenetic Regulation of Alpha-Synuclein Expression

Thaw neural progenitor cells or NPCs stored with a cryoprotectant in a heat block.

These NPCs express increased alpha-synuclein, a key factor in Parkinson's disease.

Transfer the thawed cells to a tube containing warm basal media to dilute the cryoprotectant.

Centrifuge to pellet the cells, remove the supernatant, and resuspend the pellet in neural media.

Add the cell suspension onto an ECM-coated multiwell plate and incubate to allow cell adhesion and proliferation.

Add lentiviral vectors containing guide RNA, deactivated Cas9-DNA methyltransferase, and an antibiotic-resistance gene.

Incubate to facilitate viral entry, reverse transcription of the viral RNA, integration into the host genome, and transgene expression.

The guide RNA directs dCas9 to the alpha-synuclein gene, where the methyltransferase-mediated DNA methylation reduces alpha-synuclein expression.

Replace the media with antibiotic-containing neural media to select transduced cells, while non-transduced cells undergo apoptosis.

Place a Cryovial with MD NPC in a 37 degrees Celsius heat block for two minutes and transfer the thawed cells to a 15 milliliter conical tube with 10 milliliters of warm DMEM-F12.

Centrifuge at 300 g for