Plenti c myc ddk p2a puro lentiviral gene expression vector

A plasmid containing an optimized chondroitinase ABC (CHase) sequence was generously provided by Dr. Elizabeth M. Muir^{80 (link)}. Pipe cloning was used to insert the CHase sequence into the pLenti-C-Myc-DDK-p2a-puro lentiviral gene expression vector (Origene Cat# PS100092) as previously described^{81 (link)}.
CHase lentivirus for transduction was produced by transfecting 4 × 10⁶ HEK293T cells with 6 µg pLenti-C-Myc-DDK-p2a-puro-chABC using X-tremeGENE HP DNA transfection reagent (Millipore Sigma Cat# 6366236001) as per manufacturer’s protocols. 48 h post transfection, viral supernatant was collected, centrifuged at 200 g for 10 min and filtered through a 0.45 µm Steriflip (Millipore Sigma Cat# SE1M003M00) to remove cellular debris. 5 × 10⁶ LNCaP cells were plated in six well plate and 18 h later, cells were transduced by replacing culture media with 1.5 mL of viral particle and 1.5 mL serum-free RPMI media, followed by Polybrene Infection/Transfection Reagent (Millipore Sigma Cat# TR-1003-G) to a final concentration of 8 µg/mL. Cells were re-transduced 24 h and 48 h after and CHase cells were selected with 5 µg/mL puromycin (Thermo Fisher Cat# A1113802).

pLenti-C-Myc-DDK-P2A-Puro Lentiviral Gene Expression Vector encoding KDM5A/C were purchased from OriGene Technologies (USA). Following transfection with lentiviral expression vector into 293 T cells, the supernatant containing lentivirals particles of 293 T cells were harvested and applied to PANC-1 cells for 48 h. Both shRNA vectors contained puromycin resistance as selection marker, so infected cells were successfully selected by puromycin for at least 2 weeks. The minimum concentration of puromycin (Sigma-Aldrich) for selection of PDAC cells was determined by Kill–Curve test. We also used the same method to establish CD44 knockout cell lines.
KDM5A/C Human Tagged ORF Clone were purchased from OriGene Technologies (USA). Transfection experiments were performed using Lipofectamine2000 (Invitrogen) according to manufacturer's procedures. The minimum concentration of neomycin (Sigma-Aldrich) for selection of PDAC cells was previously determined by Kill-Curve test. Stable KDM5A/C overexpressed cell lines (L3.6pl^Wt) were obtained following neomycin selection in cell culture for about 2 weeks. The efficiency of all transfections was assessed by western blot.