DISC1 Knockdown in Murine Neurons

For DISC1 knock-down in murine primary neurons, we chose a validated shRNA construct developed by Akira Sawa’s group (DISC1 RNAi #1) that has been shown to specifically decrease the amount of DISC1 in cortical neural cell cultures [8 (link),14 (link),37 (link)]. The commercial pLK0.1-puro non-mammalian shRNA control construct from Sigma Aldrich (reference: SHC002) was used as a scramble control. Lentiviruses were produced by calcium phosphate triple co-transfection of shRNA (see Table S3 and Figure S1 in Supporting Information), VSVG and ΔR8.9 constructs into 293FT packaging cells. Virus-containing medium was collected 48 h after transfection, and added (10 mL of lentiviral solution/3 × 10⁶ neurons) to the medium of primary neurons at 7 DIV. The medium was changed 24 h after infection, and incubation continued for 72 h.
In SH-SY5Y cells, DISC1 was silenced using commercial Mission^® shRNA lentiviral transduction particles (Sigma Aldrich, reference NM_018662) containing two alternative PLKO.1-Puro-CMV shRNA plasmids (Table S2 in Supporting Information). Mission^® pLKO.1-puro non-mammalian shRNA particles (reference: SHC002V) were used as control. Stable cell lines were generated for any of these constructs after selection with puromycin as previously described [37 (link)].