For genetic knockdown of PARP-1 in vivo, 50 μl of saline-formulated high-titer lentiviral shRNA specifically inhibiting PARP-1 (formulated high-titer lentiviral shRNA specifically inhibiting PARP-1, “PARP-1 shRNA”; Sigma-Aldrich) or control lentivirus (empty vector control plasmid DNA that contains no shRNA insert; MISSION pLKO.1-puro Empty Vector Control Plasmid DNA; Sigma-Aldrich) was administered 72 hours before VILI into male miR-223 knockout mice by an intratracheal injection.
Mission plko 1 puro empty vector control plasmid dna
Manufactured by Merck Group
The MISSION pLKO.1-puro Empty Vector Control Plasmid DNA is a laboratory tool designed for use in lentiviral-based gene expression and RNA interference (RNAi) experiments. It serves as a negative control for validating the specificity of experimental results obtained using MISSION lentiviral shRNA or overexpression constructs. The plasmid does not contain any shRNA or transgene, allowing it to be used as a baseline for comparison in experiments.
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Lab products found in correlation
4 protocols using mission plko 1 puro empty vector control plasmid dna
1
PARP-1 Knockdown in miR-223 Knockout Mice
2
Cloning of gls1-shRNA Lentiviral Vectors
3
Lentiviral Transduction of NRF2 shRNA in EP-MSCs
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To obtain lentiviral particles with shNRF2, HEK293T cells were seeded in 100 mm culture dishes at a density of 3 × 106 cells per dish. On the next day, the cells were transfected with lentiviral particles with either a nontargeting shRNA expression plasmid (MISSION Plko.1-puro Empty Vector Control Plasmid DNA, Sigma) or two shNRF2 expression plasmids (NFE2L2 MISSION shRNA Stock, TRC numbers: TRCN0000007555 (shNRF-1), TRCN0000007558 (shNRF-2), Sigma) with the Delta 8.9 plasmid (gag, pol, and rev genes) and VSV-G (envelope plasmid, Sigma) using Lipofectamine 2000 (Invitrogen). After 6 h of transfection, the medium was replaced. The shRNA-transfected HEK293T cells were maintained for 2 days, and then the supernatants were collected and stored at −70 °C. To knockdown NRF2 in EP-MSCs, the cells were seeded in 6-well plates at a density of 5 × 104 cells per well. After 48 h of infection, the medium was replaced with freshly prepared medium with 10 μg/ml puromycin dihydrochloride (Sigma), and the cells were maintained for 7 days. The knockdown efficiency of the selected cells was analyzed by western blot analysis. Below is a list of shRNAs targeting NRF2 used in this study.
1. shNRF2-1 (region: 3′ UTR)
5′-CCGGGCTCCTACTGTGATGTGAAATCTCGAGATTTCACATCACAGTAGGAGCTTTTT-3′
2. shNRF2-2 (region: CDS)
5′-CCGGCCGGCATTTCACTAAACACAACTCGAGTTGTGTTTAGTGAAATGCCGGTTTTT-3′
1. shNRF2-1 (region: 3′ UTR)
5′-CCGGGCTCCTACTGTGATGTGAAATCTCGAGATTTCACATCACAGTAGGAGCTTTTT-3′
2. shNRF2-2 (region: CDS)
5′-CCGGCCGGCATTTCACTAAACACAACTCGAGTTGTGTTTAGTGAAATGCCGGTTTTT-3′
4
Lentiviral Transduction of Prostate Cancer Cell Lines
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PC3, DU145 and LNCaP prostate cancer cell lines were seeded in a 96 multiwell, 35,000 cells/ well, and treated with Sh MIMIC Lenti miR-423-5p lentiviral particles (GE Healthcare Dharmacon V1SMHS_000254) at 2.5, 5, 10 and 20 MOI. Control empty backbone cells were obtained using lentiviral particles generated from the pLKO.1 Empty plasmid (MISSION® pLKO.1-puro Empty Vector Control Plasmid DNA Sigma Aldrich SHC001). Hexadimethrine Bromide (SIGMA H9268-5G) was used in complete growth media for each cell line to facilitate the infection. After 16 h incubation, infection media was removed and substituted with complete growth medium for each cell line. Puromycin (INVIVOGEN) was then added to each cell complete growth medium at a final concentration of 1 μg/ml for the selection. The lentiviral particles used to infect DU145, PC3 and LNCaP were produced according to the manufacturer’s recommendations. The lentiviral packaging mix was also purchased from Sigma (SHP001).
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