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 five minutes. Aspirate the supernatant and re-suspend the cells in two milliliters of pre warmed complete N2B2 medium. Add two milliliters of N2B27 to the cell suspension and plate two milliliters of cells in each well of the previously prepared BMM coated six well plate.

Incubate the cells at 37 degrees Celsius with 5% CO_2 overnight. When MD NPCs are at 70% confluence, transduce them by adding two micro liters of lentivirus guide RNA d-Cas9-DNMT3A vectors and gently swirl the plate. After incubating the cells at the same conditions as previously for 16 hours replace the N2B27 medium 48 hours after the transduction.

Add N2B27 with five micrograms per milliliter of puromycin