Puro blank lentivirus

In order to receive a generation of WWOX downregulation cell line, cells were transfected by Human WWOX sgRNA CRISPR/Cas9 (pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro) set and simultaneously we received control by transfecting by Scrambled sgRNA CRISPR/Cas9 Lentivirus (Applied Biological Materials Inc., Richmond, BC, Canada.). Additionally, the cell line was transfected with pLenti-GIII-CMV-GFP-2A-Puro lentivirus vector (Applied Biological Materials Inc., Richmond, BC, Canada), in two variants: a vector carrying human WWOX cDNA and a Puro-Blank Lentivirus as an experiment control (Applied Biological Materials Inc., Richmond, BC, Canada). Polybrene at a concentration of 8 μg/μL was added to the infection medium to enhanced the efficiency of lentiviral gene transduction. Both transductions were performed in a starving medium containing lentiviral particles at MOI = 2. In all vectors, a puromycin resistance gene was included and the selection was carried out with puromycin at a concentration of 1 μg/mL for four doses. Transfection efficiency was verified by RT-qPCR and western blot analysis.

The overexpressed WWOX profile was obtained using the GIPZ Lentiviral™ system (pLenti-GIII-CMV-GFP-2A-Puro) and Puro-Blank Lentivirus used as a control (Applied Biological Materials Inc.). Transduction was performed in starving medium containing 8 µg/mL polybrene and lentiviral particles at MOI = 3. After 24-h transduction, the medium was changed for full medium. After 72-h, antibiotic-based clone selection (puromycin, 1 µg/mL) was performed.
For downregulated WWOX profile, sgRNA CRISPR/Cas9 (pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro) was used, with Scrambled sgRNA CRISPR/Cas9 (Applied Biological Materials Inc.) as control. Transduction was performed in starving medium containing 8 µg/mL polybrene and lentiviral particles at MOI = 2. Transduction medium was changed into full medium after 24-h of incubation. This was followed by antibiotic-based clone selection (puromycin, 1 µg/mL) after 72-h.

The GIPZ™ lentiviral system (pLenti-GIII-CMV-GFP-2A-Puro) was chosen to overexpress WWOX, with Puro-Blank Lentivirus as control (Applied Biological Materials Inc., Richmond, BC, Canada). The cells were transduced in starvation medium with 8 µg/mL polybrene as the vehicle (Merck Life Sciences Sigma Aldrich, Darmstadt, Germany) and lentiviral particles at multiplicity of infection (MOI) = 1.5. After 24 h of incubation, the viral medium was changed to a complete medium. Antibiotic-based clone selection was performed using 1 µg/mL puromycin (Merck Life Sciences Sigma Aldrich, Darmstadt, Germany) after 72 h. The efficiency was confirmed by Western blot analysis.