Lenti x concentrator

HEK293T cells were grown to 60% confluency in 225cm² flasks. To proceed with viral packaging, DMEM was removed and replaced with 13 mL of pre-warmed OptiMEM (ThermoFisher #31985). The pMD2.G, psPAX2, and pooled library plasmids were combined with Lipofectamine PLUS reagent (ThermoFisher #15338) and OptiMEM. The combined solution was added and incubated for 6 h, when OptiMEM was replaced with media supplemented with 1% bovine serum albumin (ThermoFisher #A9647). After 60 h, media was harvested, centrifuged at 3000 rpm for 10 min at 4 °C, filtered through a 0.45um filter (VWR #28145), and stored at 4 °C overnight in Lenti-X concentrator (Clontech #631231) at a 3:1 virus to Lenti-X concentrator volume ratio. The following day the virus was centrifuged at 1500×g for 45 min at 4 °C and the supernatant was poured off. The viral pellet was resuspended in DMEM culture media at one-tenth the original volume and stored at -80 °C. Packaging of all vectors was performed identically to the pooled library, with reagents scaled down proportionally according to the surface area of the flask.
Viral packaging of single sgRNAs used for validation was performed in 6-well tissue culture treated plates. The protocol was performed as above except cells were 90% confluent, the incubation after transfection was reduced to 48 h and virus was either frozen as before, or in some cases used fresh.

Cell culture based hepatitis B virus (HBVcc subtype adw2) was prepared from freshly collected supernatants of the HBV stably transfected HepG2 cell line, clone 2.2.15 [22 (link)]. HepG2.2.15 cells were maintained in DMEM (Hyclone, GE Healthcare), 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin. The supernatant was collected at 7 d after adding 380 µg/mL G418 and concentrated 100-fold. The collected supernatants were filtered through 0.45 µm and viral particles were concentrated by Lenti-X™ Concentrator (Clonetech, Takara Bio, Mountain View, CA, USA). The mixtures were incubated at 4 °C at least 30 min before centrifugation at 1500× g for 45 min at 4 °C following the manufacturer’s instructions. The supernatants were removed and the pellet was gently resuspended with FBS at dilution 1:100 of original volume of supernatant. 100× HBV was aliquoted and stored at −80 °C until use. The HBV titer was about 6.28 × 10⁸ HBV genome equivalents/mL using the serial dilutions of a known amount of plasmid HBV 1.3-mer WT replicon (Addgene plasmid # 65459) as a standard curve.

Overexpression of SKP2 was performed by lentivirus mediated transduction using standard protocols. Briefly, virus particles were generated using HEK293T cells (ATCC) transfected with CMV-SKP2 or CMV-GFP lentivirus transfer plasmids (a kind gift from Dr. Liang Zhu, Albert Einstein College of Medicine) and packaged with PMD2.G and pSPAX2 (Addgene). Virus-containing supernatant was collected between 24 and 72 hours post-transfection and concentrated with Lenti-X concentrator (Clonetech) according to the manufacturer’s suggested protocol. 143B and SaOS-2 cells were then transduced by addition of either GFP control or Skp2 particles in presence of 8 µg/mL polybrene, and expression of the target proteins was confirmed by fluorescence microscopy and qRT-PCR. The resulting cell lines were used for downstream assays without further modification.

Takara (Clonetech) Lenti-X Packaging Single Shots (4^th generation) were used following manufacture protocol. Supernatant was collected 48–72 hours post-transfection to infect cells (with 10μg/mL polybrene). For the timepoints experiment, when concentrated infection is indicated, Lenti-X concentrator (Clonetech) was used to concentrate the virus.

Lentivirus supernatant was produced by co-transfection of pLVX-IRES-Puro [Empty Vector (EV)] or pLVX-3Flag-CEP164-IRES-Puro (CEP164) with Δ8.9, pcRev, and VSVG plasmids (gift from M. Hagiwara) into Lenti-X 293T cells using PEI Max. The virus supernatant was harvested 72 h post-transfection and concentrated using Lenti-X Concentrator (Clonetech). Panc1 cells were incubated with virus in the presence of 5 μg/ml polybrene (Nacalai Tesque) for 72 h. The infected Panc1 cells were subsequently cultured in medium with 5 μg/ml puromycin (Nacalai Tesque) for 8 days. Established cells (WT + EV, Cep164-1 + EV, and Cep164-1 + Cep164) were cultured in medium with 3 μg/ml puromycin.