Psilencer1.0 u6 sirna expression vector

For knockdown of the α2δ1 subunit, siRNA target sequences corresponding to the α2δ1 coding region (CACNA2D1, GenBank accession number NM_009784.2) (Obermair et al., 2005 (link)) were selected and tested for efficient knockdown. The siRNA was expressed as shRNA under the control of a U6 promoter (derived from the pSilencer1.0-U6 siRNA expression vector, Ambion) cloned into the pβA-eGFP plasmid (Obermair et al., 2010 (link)). For lentiviral expression, α2δ1 shRNA was cloned into pHR as previously described (Subramanyam et al., 2009 (link)). For knockdown of the α2δ3 subunit, four 29mer shRNA constructs against rat Cacna2d3 (Gene ID 306243) cloned in lentiviral GFP vector (pGFP-C-shLenti Vector, catalog #TR30023) were ordered from OriGene Technologies (catalog #TL713428). Based on their specificity for rat and mouse α2δ3, two of these constructs were tested for their knockdown efficiency where the construct “C” was evaluated to results in a reduction of α2δ3 expression down to 40%-50%. As control for α2δ-knockdown experiments, a noneffective 29-mer scrambled shRNA cassette cloned into pGFP-C-shLenti Vector (catalog #TR30021; OriGene Technologies) was used.

The transcriptional unit for NPM1 knockdown was amplified from a pSilencer1.0–U6 siRNA Expression Vector (Ambion, Austin, TX, USAUSA) containing a shRNA sequence against the 3′UTR region of NPM1 isoform 1 (TRCN0000062268, NPM1 MISSION shRNA, Sigma). The PCR product was then digested and inserted into XbaI restriction site of the lentiviral transfer plasmid pLGW [70 (link)]. This plasmid includes a GFP reporter gene under the SV40 promoter, and HIV-based third-generation lentiviral vector elements to produce infective particles once co-transfected with packaging plasmids pLP1, pLP2 and pLP/VSVG in HEK293T cells (ViraPower Lentiviral Packaging Mix, Thermo Fisher Scientific, Waltham, MA, USA). Virus production and subsequent titration of viral particles were performed according to previously described protocols [71 (link)]. HPB-ALL cells were infected by spinoculation at a 10x multiplicity of infection (MOI10) [72 (link)]. Briefly, the cells were seeded as 0.5 × 10⁶ cells/mL, in preconditioned medium, with polybrene (8 µg/mL) plus the viral supernatant, and then centrifuged at 800× g for 30 min at 32 °C. On the next day, fresh complete medium was added and the cells allowed to recover for an additional 48 h. HPB-ALL cells were evaluated by flow cytometry analysis for confirmation of GFP expression as a measure of transduction efficiency (Cyflow, Partec, Germany).