Global ultrarapid lentiviral titer kit

The human pull of shRNA NSun2 and scramble shRNA control were obtained from Applied Biological Materials Inc. To prepare lentivirus for in vitro experiments, the second-generation packaging system (which generates replication-deficient lentivirus) was used for all experiments [99 (link)]. Packaging vectors such as psPAX2 and pMD2.G were obtained from Addgene. Briefly, lentiviral constructs for shRNA or scramble control were co-transfected with the packaging vectors into HEK293T cells using CalFectin (SignaGgen). Supernatants containing virus were collected 48 h after transfection. After centrifugation at 1000 rpm for 10 min, the supernatants were passed through a 0.45 μm PDVF filter unit (Nalgene). The viruses were concentrated 20–30 × by centrifugation in an Amicon Ultra centrifugal filter (100 K) (Millipore) following the manufacturer's instructions. Lentiviral stock titration was carried out using the Global UltraRapid Lentiviral Titer Kit (System Biosciences). Viruses were aliquoted and stored at − 80 °C.

The pMIRNA1 plasmid and pPACKH1 lentivector packaging kit were purchased from SBI (System Biosciences, Menlo Park, CA, USA). A 500‐bp DNA fragment flanking pre‐miR‐21 was inserted downstream of the CMV promoter in pMIRNA1 to generate pMIRNA1‐miR‐21. Viral packaging was performed according to the manufacturer's instructions. Virus particles (lenti_miR‐21 and lenti_GFP) were harvested and concentrated using PEG‐it Virus Precipitation Solution (SBI). Virus titer was determined in 293TN cells using a global ultrarapid lentiviral titer kit (SBI). For gene transduction into PATU8988 and PANC‐1 cells, the recombinant virus particles were added to the culture medium of the cells at an MOI = 3–5.

Lentiviruses were prepared using System Biosciences lentivirus technology. Two-hundred ninety-three TN cells (System Biosciences (SBI) LV900A-1) were seeded in 150-mm plates at 7.5 × 10⁶ cells per plate with 20 mL DMEM without antibiotics for 24 h. Cells were then transfected with 22.5 μg pPACKH1 HIV packaging mix (SBI LV500A-1) and 4.5 μg of pCDH lentiviral vector using BioT transfection reagent. Viral supernatant was collected 48 and 72 h post-transfection. After centrifugation for 15 min at 3000×g to remove debris, 1× PEG-it (SBI LV810A-1) was added to precipitate virus. After incubation at 4 °C for at least 16 h, centrifugation (30 min at 1500×g) was used to collect viral particles. Virus was resuspended in a small volume (300–500 μL) 1× phosphate buffered saline (PBS) and titered using the Global UltraRapid Lentiviral Titer Kit (SBI LV961A-1). Transduction of target cells was performed according to manufacturer’s protocol (SBI). Briefly, cells were seeded at 1.0 × 10⁵ cells per 12-well or 2.5 × 10⁵ cells per six-well plate. After 24 h, cells were treated with fresh media, 1× TransDux (SBI LV850A-1), and lentivirus at a multiplicity of infection (MOI) of 10. MOI was determined using previously published methods. Infected cells were collected for subsequent assays after 72 h.

Wild-type H-1PV was produced, purified and titrated as previously described^{27 (link)}. Recombinant H-1PV (recH-1PV-EGFP) harbouring the EGFP-encoding gene was produced according to the protocol described in El-Andaloussi et al.^{55 (link),59 (link)}. Lentivirus expressing LAMC1-specific guide RNA (lentiCRISPRv1-sgLAMC1) was produced as previously described^{60 (link)}. In brief, HEK293T cells were transfected with lentiCRISPRv1-sgLAMC1, psPAX2 (Addgene, Cambridge, MA, USA) and pMD2.G (Addgene) plasmids at the ratio (2:1.5:1) using the Lipofectamine LTX and Plus reagent (Life Technologies Europe, Bleiswijk, Netherlands) according to the manufacturer’s protocol. At 70 h post transfection, the culture medium containing lentiviral particles was removed and centrifuged, and cell debris was discarded. The supernatant was filtered through a 0.45 µM filter (Millipore Steriflip HV/PVDF; Merck Millipore, Burlington, MA, USA), concentrated 100× with PEG-it (BioCat Gmbh, Heidelberg, Germany) and re-suspended in DMEM medium containing 10% FBS, 2 mM l-glutamine and 1% bovine serum albumin. Lentivirus aliquots were stored at −80°C. Lentivirus titration was carried out using Global UltraRapid Lentiviral Titer Kit (SBI System Biosciences, Palo Alto, CA, USA).

The pMIRNA1 plasmid and pPACKH1 lenti_vector packaging kit were purchased from SBI (System Biosciences, USA). A 500 bp DNA fragment flanking miR-320a was inserted into the downstream of CMV promoter in pMIRNA1 to generate pMIRNA1-miR-320a. Viral packaging was performed according to the manufacturer’s instructions. Virus particles (lenti-miR320a and lenti_GFP) were harvested and concentrated using PEG-it Virus Precipitation Solution (SBI). Virus titer was determined in 293TN cells using global ultrarapid lentiviral titer kit (SBI). For gene transduction into PATU8988 and PANC-1 cells, the recombinant virus were added to the culture medium of the cells as MOI = 3~5.