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

Samenvatting

Source: Tagliafierro, L., et al. Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models. J. Vis. Exp. (2019)

This video demonstrates the lentiviral transduction of neural progenitor cells (NPCs) for targeted epigenetic modification of alpha-synuclein, a key protein in Parkinson’s disease. The protocol enables methyltransferase-mediated DNA methylation, reducing SNCA expression for studying gene regulation and potential therapeutic interventions.

Protocol

1. Differentiation of dopaminergic neural progenitor cells

1. Thawing MD NPCs (midbrain dopaminergic neural progenitor cells)
   1. Prepare a BMM (basic matrix membrane)-coated plate and warm complete N2B27. Add 10 mL of warm DMEM/F12 to a 15 mL conical tube. Place a cryovial in a 37 °C heat block for 2 min.
      1. Transfer the cells from the cryovial to the tube containing DMEM/F12. Centrifuge at 300 x g for 5 min.
      2. Aspirate the supernatant, resuspend the cells in 2 mL of N2B27, and add the cell suspension to one well of the BMM-coated plate. Incubate the cells at 37 °C with 5% CO₂.

2. Transduction of MD NPCs and the analysis of methylation changes

1. Transduction of MD NPCs
   1. Transduce MD NPCs at 70% confluence with LV (lentiviral vector)-guide RNA/deactivated Cas9-DNMT3A (DNA methyltransferase enzyme 3A) vectors at MOI (multiplicity of infection) = 2. Replace the N2B27 medium 16h posttransduction.
   2. Add N2B27 with 5 µg/mL puromycin, 48 h after the transduction. Culture the cells for 3 weeks in N2B27 plus puromycin to obtain the stable MD NPC lines. Cells are ready for downstream applications (DNA, RNA, protein analyses, phenotypic characterization, freezing, and passaging).

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Equipment
15 mL conical centrifuge tubes	Corning	430791
6-well plates	Corning	3516
Allegra 25R tabletop centrifuge	Beckman Coulter	369434
Inverted fluorescence microscope	Leica	DM IRB2
Cell culture reagents
Antibiotic-antimycotic solution, 100X	Sigma Aldrich	A5955-100ML
B-27 Supplement (50X), minus vitamin A	Thermo Fisher Scientific	12587010
Corning Matrigel hESC-Qualified Matrix	Corning	08-774-552
DMEM-F12	Lonza	12-719
N-2 Supplement (100X)	Thermo Fisher Scientific	17502001
Neurobasal Medium	Thermo Fisher Scientific	21103049