Lenti pac hiv expression packaging kit

For lentivirus production, the Lenti-Pac 293Ta packaging cell line was transfected with various lentivirus expression plasmids using the Lenti-Pac HIV Expression Packaging Kit (GeneCopoeia, Inc., Rockville, MD, United States). Lentiviral supernatants were used to infect 3 × 10⁶ B cells, which were stimulated with 5 μg/ml CpG ODN (Class B, invivoGen, San Diego, CA, United States). Two days after infection, the GFP-positive population was sorted out using the FACsAria Cell Sorter (BD Biosciences Immunocytometry Systems). The resulting data were analyzed on a BD LSRFortessa^TM cell analyzer using FlowJo software 6.0 (TreeStar Inc., Ashland, OR, United States).
After 48 h of incubation in CpG ODN, the supernatants were harvested, and ELISA was carried out using a commercial kit (NeoBioscience, China). The proliferation of sorted GFP+ virally infected cells was measured by CFSE assays as previously described (Aab et al., 2017 (link)).

Lentivirus infection was performed using Lenti-Pac™ HIV Expression Packaging Kit (GeneCopoeia, Guangzhou, China). Lentiviruses produced in 293 T cells were used to infect BC cells cultured in a medium containing 5 μg/mL polybrene. Lentiviral vectors expressing four independent shRNAs against PDEF or AR and those inducing PDEF or MAD1 overexpression were obtained from GeneCopoeia. After the infection, cells were selected using puromycin.

A lentiviral open reading frame plasmid Lv-TK1 (EX-C0529-Lv105) containing human TK1 mRNA complete sequence [PubMed cDNA clone MGC number: 3644] and a control plasmid (EX-NEG-Lv105) were obtained from GeneCopoeia, USA. The sequences of cloned plasmids were confirmed by DNA sequencing using 5’-GCGGT AGGCG TGTAC GGT and 5’-ATTGT GGATG AATAC TGCC as the forward and reverse primers, respectively. Pseudo lentiviral particles for TK1 overexpression and the control were produced with Lv-TK1 or the control plasmid and Lenti-Pac HIV expression packaging kit (GeneCopoeia, USA) on 293T cells according to manufacturer’s protocol. Pseudovirus titer was estimated on Hep3B cells under the selection of puromycin (0.5 μg/mL) as 1.6 × 10⁷ and 1.2 × 10⁷ transducing units/mL for TK1 and the control, respectively. Transduction of HL-7702 and Bel-7402 cells was carried out at a cell density of 50,000 in a 24-well plate with 80 μL pseudovirus stock solution plus poloxamer F108 (100 μg/mL, 10 μL) and polybrene 100 (100 μg/mL, 10 μL). After 24 h, the transduction media were replaced with the normal DMEM growth media, and cells were grown in a 6-well plate for 7 days. Western blot analysis and cell viability MTT assay with compound treatment were then carried out similarly as described in siRNA study.

Four short hairpin RNA (shRNA1-4), targeting different regions of Arhgef11 were cloned into Lenti-Pac HIV Expression Packaging Kit (GeneCopoeia, MD). All four shRNA were specific to rat and one (of four) was a perfect match with human Arhgef11 (S2 Fig). Plasmids containing each shRNA/GFP or scrambled control and packaging plasmid were co-transfected into HEK293T/17 or NRK (~ 90% confluent) at ratio of 2:1:1 per manufacture instructions. Cells for GFP control plates were checked regularly under the fluorescent microscope. Culture medium containing virus was collected 48–72 hrs after transfection, harvested, and concentrated 10-50-fold using Lenti-Pac Lentivirus Concentration Solution (GeneCopoeia, MD). Virus titers were determined using H1299 cells by fluorescence analysis. 10^9 IU/ml virus was stored in 1 ml aliquots at −80 °C until cell transduction. HEK293T and NRK were transduced with concentrated lentiviral stocks in the presence of 8 μg/ml polybrene (Sigma-Aldrich, MO). Stably transduced cells were selected using 2ug/ml puromycin (Life Technologies l) for 3–5 days and purity of transduced cells was evaluated by GFP fluorescence microscopy.

The PAK6 shRNA were packaged using HEK 293 T cells by Lenti-Pac HIV Expression Packaging Kit (GeneCopoeia) and then infected into AGS and BGC-823 cells. The PAK6, CS-HR-EGFP-M68, and CS-SceI-M23 expression constructs were transfected into HEK 293 T cells using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA).
Further, the lentivirus expressing PAK6 were infected into MGC-803 and SGC-7901 cells. Stable cell lines were selected with the presence of 500 ug/ml G418 (Biyuntian, China), 100 ug/ml hygromycin (Biyuntian, China), or 4 ug/ml puromycin (Biyuntian, China) according the relative resistance genes of constructs. Details about the products are showed in the supplementary materials.

Transfections were performed with the Lenti-Pac HIV Expression Packaging Kit (GeneCopoeia, Rockville, MD, USA). For generating lentiviral particles, packaging plasmids (VSVG and ΔR) and expression plasmids (KD and KD/SCR) were transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). The cells are then infected with a lentivirus to produce stable SVEP1 KD or KD/SCR cells. Six-well plates placed with 2 × 10⁵ cells were infected by lentivirus for 6–8 h. Then, added equal volume of 10% FBS DMEM and the transfected cells were cultured for another 48 h. The stably transfected cell line was obtained under the puromycin (GIBCO) selection about 7 days.