Construction of the lentivirus vector for PPP5C short hairpin (sh)RNA and virus packaging

Based upon the sequence of PPP5C ({"type":"entrez-nucleotide","attrs":{"text":"NM_001204284.1","term_id":"324021715","term_text":"NM_001204284.1"}}NM_001204284.1), a responsible shRNA sequence (5′-GAGACAGAGAAGATTACAGTACTCGAGTACTGTAATCTTCTCTGTCTCTTTTT-3′) was generated to target PPP5C and a control shRNA sequence (5′-TTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3′) was designed and synthesized. The recombinant vectors, PPP5C shRNA (shPPP5C) and control shRNA (shCon), were designated to carry the corresponding shRNA. T4 DNA ligase (New England BioLabs, Inc., Ipswich, MA, USA) was used to construct shRNA fragments (50 ng), which were cloned into the lentiviral expression vector pGP (Shanghai Hollybio, Shanghai, China) prior to being digested by EcoRI and BamHI (New England BioLabs, Inc.). Lentivirus was generated by co-transfection of HEK293 cells with recombinant vectors and packaging plasmids (pVSVG-I and pCMV ∆ R8.92; Shanghai Hollybio). The supernatants were collected 96 h after transfection to extract the lentivirus that may express PPP5C shRNA or control shRNA. The lentivirus was then purified via ultracentrifugation in a condition of 100,000 × g for 30 min at 4°C. PANC-1 cells were infected with the concentrated virus at a multiplicity of infection of 10 and mock-infected cells were used as negative controls. Since the lentivirus carries a green fluorescence protein (GFP) as a reporter and this GFP is driven by the cytomegalovirus promoter, the titer of lentivirus was determined by counting the number of cells that expressed GFP under fluorescence microscopy under ×100 magnification (Olympus Corporation, Tokyo, Japan) following 48 h of infection. The efficiency of PPP5C-knockdown was subsequently measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis.