Mission shrna lentiviral particles

HEK293 T-Rex cells were purchased from Invitrogen. C5 cells contain a bidirectional artificial heat shock promoter with 8 times mulitmerized HSEs driving the expression of firefly luciferase and GFP and were previously described [27 (link)]. XshHSF1-5-13 HSF1 knock-down cells were generated by lentiviral transduction of HEK293 T-Rex cells with MISSION shRNA lentiviral particles (TRCN0000318712, Sigma Aldrich) and single clone selection. All cell lines were kept in an incubator at 37°C with 5% CO₂ in Dulbecco’s modified eagle medium (DMEM) with 4.5 g/L glucose, Na-Pyruvate L-glutamic acid, 10% fetal bovine serum and 1 x Penicillin / Streptomycin. DMEM containing all the before mentioned substances will be called DMEM complete hereafter. For experiments in 96-well plates, the plates were coated with polyethyleneimine (PEI) [31 (link)] before seeding. Transient transfections were conducted as described in [28 (link)]. For microscopy cells were seeded in a 6-well plate and treated with cadmium in the same way as for luciferase reporter assays. After 48 h cells were stained with PI (1 ng/μL) for 30 min before imaging with a Zeiss Observer microscope. P-values were calculated by the Student’s t-test. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

For stable gene knockdown, the MISSION shRNA lentiviral particles from Merck/Sigma Aldrich (St. Louis, MO, USA) were used. Briefly, 8 × 10³ of K562 cells were cultured in 96-well plates, incubated for 2hat 37°C, and then 5 different lentiviral particles were added at MOI of 5. After incubation of cells for 72hat 37°C, 1 μg/mL puromycin was added. To establish stable knockdown cell lines, clones 1–5 were subjected to selection for additional 14 days in medium containing 1 μg/mL puromycin and pooled. The level of gene silencing was verified with RT-qPCR and Western Blot analysis. MISSION shRNA lentiviral particles used: MISSION TRC2 pLKO.5-puro Non-Mammalian shRNA Control Transduction Particles (SHC202V); MISSION shRNA Lentiviral Transduction Particles for FMRP knockdown (SHCLNV-NM_002024): TRCN0000059759 (clone 1), TRCN0000286973 (clone 2), TRCN0000286972 (clone 3), TRCN0000294378 (clone 4), TRCN0000298271 (clone 5); for TIAR knockdown (SHCLNV-NM_003252): TRCN0000276257 (clone 1), TRCN0000276212 (clone 2), TRCN0000276240 (clone 3), TRCN0000276211 (clone 4), TRCN0000017212 (clone 5).

To knock-down CD74 expression, BJAB and Raji cells were transduced using MISSION shRNA lentiviral particles targeting CD74 or non-target (NT) controls, according to manufacturer’s protocol (Sigma-Aldrich), and kept under selection with 1.9 μg/mL of puromycin.
The shRNA sequences from MISSION shRNA lentiviral particles were inserted between BamHI and EcoRI restriction sites in the multiple cloning cassette of pSIREN-Shuttle vector (Clontech). The U6 promoter-shRNA cassettes were then recloned to KpnI and ApaI sites of the pEPI-1 vector kindly provided by Dr. A. C. Jenke (HELIOS Children's Hospital Wuppertal, Germany) [28 (link)]. The correct sequences of inserted shRNAs were confirmed by sequencing (SeqWright).
Obtained plasmids were transfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s recommendations. Transfected cells were sorted 48 h post transfection based on green fluorescent protein (GFP) expression, using a flow-activated cell sorter BD FACSAria II (BD Biosciences) followed by limited dilutions and selection with geneticin (1350 μg/mL; Invitrogen) for 2 weeks.

Pathogen free conditions were maintained through the lifetime of twelve male BALB/c nude mice (4 weeks old). The approval of xenograft in vivo assay was obtained from Zhongshan hospital. Firstly, overexpression of NDRG2 (Lv-NDRG2 OE) was performed using MISSION® shRNA lentiviral particles (Sigma-Aldrich), which were designed to overexpress the production of NDRG2 in SKOV3 cells. The cultures that were transfected with lentiviral particles with negative control vector were used as the control group (Lv-Vector). Subsequently, Lv-NDRG2 OE or Lv-Vector transfected SKOV3 cells (1 × 10⁶) were subcutaneous injected into the armpit of nude mice respectively. Caliper was adapted in measuring tumor volume following the length×width²/2 formula. The average volume of tumor was measured for 3 times every 3 days. At the termination of the experiment (the 25th day), mice were killed and the tumor was excised from each mouse to measure the average volume and weight.

Six anti-Brca1 shRNAs were obtained from Sigma-Aldrich (Mission shRNA lentiviral particles, parental vector on pLKO.1-purobackbone). Their ability to reduce BRCA1 protein levels was assessed in primary mouse neuronal cultures 1 week after infection. The sequences of the two most effective shRNAs (sh1 and sh2) were: sh1: 5′-CCACAGGTAAATCAGGAATTT-3′ (ref. 15 (link)) and sh2: 5′-GTGCTTCCACACCCTACTTAC-3′. The sh1 sequence is in the coding sequence of BRCA1 mRNA, and the sh2 sequence is in the 3′-untranslated region. The sequence of the control, scrambled shRNA was 5′-CCACTACCGTTGTTATAGGTG-3′. Each sequence was inserted into the FUGW plasmid backbone and used to generate lentiviral vectors as described6 (link)62 (link). Lentiviruses were bilaterally injected stereotaxically into the DG as described6 (link), using 1.5 × 10⁶ titre unit (TU) per DG. No specific method of randomization was used to determine the treatment of each mouse. GFP expression was used as an indication of viral transduction. Note, however, that the GFP intensity does not reliably reflect the abundance of shRNA expressed in transduced cells, as the promoters driving the expression of these transgenes are activated by different RNA polymerases: GFP expression is RNA polymerase II dependent, whereas expression of the shRNA is RNA polymerase III dependent.

MISSION shRNA lentiviral particles were purchased from Sigma-Aldrich. Transduction of HaCaT cells with lentiviral-based shRNAs targeting NRF2 (SHVRS-NM_006164) or scrambled nontarget negative control (SHC002V) was performed as previously described [36 (link)]. Transducted cells were maintained in DMEM medium containing 1.0 μg/ml puromycin (Invitrogen).

SH-SY5Y cells were transduced using Mission shRNA lentiviral particles (Sigma) with a MOI of 15. The lentivirus coded either for a non-targeted (nt) shRNA (SHC002V) or an shRNA directed against PINK1 (TRCN0000007101). For selection of successfully transduced cells Puromycin (final concentration 1 µg/ml) was used. After 14 d the PINK1 mRNA expression of transduced cells was 50% reduced in comparison to the nt cells. Therefore, PINK1 knockdown cells were subjected to one round of subcloning. This procedure resulted in two clones with stronger reduction of PINK1 expression but only one of them demonstrated a stable downregulation and was therefore used for the experiments.