The pLenti-C-Myc-DDK and pLenti-C-Myc-DDK-UBE2N plasmids were purchased from OriGene (Rockville, USA). Lentivirus overexpressing UBC13 and control lentivirus were produced with lentiviral packaging kits (OriGene). At 24 and 48 h after transfection, supernatants containing lentivirus were collected, filtered, aliquoted, and stored at −80 °C. FLT3L-expanded BMDCs were transduced with lentivirus in the presence of 4 μg/ml polybrene (Sigma-Aldrich). The transduction mixture was centrifuged at 1000 × g at 32 °C for 90 min and further incubated in the cell incubator for 5 h. Thereafter, the transduced cells were incubated in normal BMDC culture medium for 3 days.
Lentiviral packaging kit
Manufactured by OriGene
Sourced in China, United States
The Lentiviral Packaging Kit is a laboratory tool used for the production of lentiviral particles. It contains the necessary plasmids and reagents required to generate recombinant lentiviral particles for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Merck Group (3 mentions)
Lentiviral gfp vector, by OriGene (2 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Sh nc, by Merck Group (1 mentions)
Shduxap8, by GenePharma (1 mentions)
Plko 1 puro, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Sh nc, by GenePharma (1 mentions)
Mimic nc, by GenePharma (1 mentions)
Hexadimethrine bromide, by Merck Group (1 mentions)
Ps100064, by OriGene (1 mentions)
Plenti ddk p2a puro empty vector, by OriGene (1 mentions)
Puromycin, by Merck Group (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Skov3, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
11 protocols using lentiviral packaging kit
1
Lentiviral Overexpression of UBC13 in BMDCs
2
Lentiviral-Mediated NR4A3 and Bnip3 Knockdown
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For lentiviral based endogenous NR4A3 and Bnip3 gene knockdown, three NR4A3 shRNA and plasmid DNA constructs in hU6-MCS-CBh-gcGFP-IRES-puromycin-shRNA lentiviral vectors complementary to rat NR4A3 gene coding sequences were purchased from Genechem (Shanghai, China). The shRNA sequences are 5'-CCGGGCAGACTTATGGCTCGGAATACTCGAGTATTCCGAGCCATAAGTCTGCTTTTT-3', 5'-CCGGCCTCCGATCTGTATGATGAACCTCGAGATTCATCATACAGATCGGAGGTTTTT-3' and 5'-CCGGCGGCCTTTGATCAAGATGGAACTCGAGTTCCATCTTGATCAAAGGCCGTTTTT-3'. The Bnip3 Rat shRNA Lentiviral Particle (CAT#: TL711845V, OriGene) and Lentiviral Packaging Kits (CAT#: TR30037) were purchased from OriGene (Beijing, China). The recombinant shRNA lentiviral plasmid or scrambled shRNA control vector was transfected into HEK-293 cells to generate lentiviruses. Thereafter, shRNA lentiviruses were transduced into to NRVM.
Adenoviral carrying full-length, three truncated (ΔAF1, ΔDBD, and ΔLBD) and two mutant (S376D, S376A) human Nr4a3 cDNA clones were constructed, packed, and puri ed by GeneChem (Shanghai, China). NRVM cells were transfected with Adv-Nr4a3 cDNA clones.
Adenoviral carrying full-length, three truncated (ΔAF1, ΔDBD, and ΔLBD) and two mutant (S376D, S376A) human Nr4a3 cDNA clones were constructed, packed, and puri ed by GeneChem (Shanghai, China). NRVM cells were transfected with Adv-Nr4a3 cDNA clones.
3
Overexpression and Knockdown Protocols for miR-485-5p and Regulatory Genes
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shDUXAP8, shNC, human miR-485-5p mimic, and mimic NC were purchased from GenePharma (Shanghai, P.R. China). shDUXAP8 or shNC sequences were cloned into lentiviral vector pLKO.1-puro (Sigma, St. Louis, MO, USA). To produce lentivirus, lentiviral vector was co-transfected into HEK293T cells with packaging vectors using the Lentiviral Packaging Kit (OriGene, Rockville, MD, USA) according to the manufacturer’s protocol. After 48 h, lentivirus-containing supernatant was harvested from the culture, centrifuged at 500 × g for 5 min to remove cell debris, and applied to target cells in the presence of polybrene (8 μg/mL; Sigma) overnight. Then, target cells were cultured in fresh complete growth medium for 48 h and selected in medium containing puromycin (5 μg/mL) for a further 10 days.
To overexpress FOXM1 or FUS, the human FOXM1 or FUS cDNA sequence was cloned into pcDNA3.1 vector (Thermo Fisher Scientific, Waltman, MA, USA). The empty vector was used as the NC. The transfection of pcDNA3.1 vector or miRNA mimics was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.
To overexpress FOXM1 or FUS, the human FOXM1 or FUS cDNA sequence was cloned into pcDNA3.1 vector (Thermo Fisher Scientific, Waltman, MA, USA). The empty vector was used as the NC. The transfection of pcDNA3.1 vector or miRNA mimics was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.
4
Generating HOTAIR Overexpressing Cells
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The sequence of HOTAIR (NR_003716) was cloned from the LZRS-HOTAIR plasmid (Addgene plasmid #26110) and inserted into the pEGFP-Lv105 vector (Capital Biosciences) as the working HOTAIR plasmid. pEGFP-Lv105 empty vector was used as a control. Then, we applied lentiviral packaging kit (Cat#3D5F03, Origene) in HEK293T cells to produce lentiviral particles. The MDA-T41 cells were transduced with HOTAIR plasmid- or empty vector-lentiviral particles in 8 µg/ml hexadimethrine bromide (Sigma)-containing growth medium to generate MDA-T41 HOTAIR-OE cells or Control cells, respectively. Then, cells were revived in normal growth medium for an additional 24–28 h with subsequent selection in puromycin-containing growth medium.
5
Lentiviral Overexpression and Knockdown
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Lentiviral GFP vectors containing NeuroD1 shRNA or scrambled shRNA were purchased from Origene (Rockville, MD, USA). MiR-30a-5p and miR-153-3p mimics were synthesized by Genolution (Seoul, Korea). 293T cells were co-transfected with NeuroD1 shRNA, scrambled shRNA, and miRNAs and packing plasmids using Lentiviral packaging kit (Origene) according to the manufacturer’s instructions. After 48 h and 72 h of transfection, culture supernatants containing lentiviral particles were harvested. Then, BV-2 cells and primary cells were infected with the harvested lentiviral particles.
6
Lentiviral Transduction of EGFR Variants
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The EGFR cDNA clones, including WT EGFR, L858R EGFR, L858R/T790M EGFR, and Del E746-A750 EGFR were subcloned into the lentivirus vector (PS100064, Origene) by SgfI and MluI. Lentiviral Packaging Kit (TR30037, Origene) was used for virus package in 293T cells. The Ba/F3 cells were infected with lentivirus together with 8 μg/mL polybrene for 24 h. 48 h later, 1 μg/mL puromycin was added to the cell culture medium and maintained for another 7 days for the selection of stable cell lines.
7
Overexpression of Human PGC-1α
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The isoform 1 of human PGC-1α (NM_001330751) was cloned by PCR into the pLenti-DDK-P2A-Puro empty vector (OriGene Technologies, PS100092). Primer sequences are reported in Additional file 1 : Primer List. Lentivirus particles containing either the empty plasmid or the PGC-1α plasmid were prepared using the Lentiviral Packaging Kit (OriGene, TR30037) following manufacturer’s instructions and used to infect the cell lines. In cybrids, stable PGC-1α overexpression has been performed selecting positive clones by puromycin resistance. Differently, transient PGC-1α overexpression has been carried out in fibroblasts, which have been analyzed after 72 h from the infection.
8
Generating stable EGFR mutant cell lines
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EGFR cDNA clones, including WT EGFR, L858R EGFR, L858R/T790M EGFR, and Del E746-A750 EGFR, were subcloned into the lentivirus vector by SgfI and MluI. The Lentiviral Packaging Kit (TR30037, Origene) was used for virus package in 293 T cells. The Ba/F3 cells were infected with lentivirus together with 8 μg/mL polybrene for 24 h. Two days later, 2 μg/mL puromycin was added to the cell culture medium and maintained for another 7 days for stable cell selection.
9
Lentiviral Overexpression of NQO1 in Cybrids
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Lentivirus particles containing a pLenti-C-Myc-DDK-P2A-Puro plasmid empty (OriGene, PS100092) or with human NQO1 isoform 1 (OriGene, RC200620L3) were prepared using the Lentiviral Packaging Kit (OriGene, TR30037), following manufacturer’s instructions and used to infect the cybrid cell lines (H42A and RJ206). Infected cells were selected with 0.25μg/mL and 0.75μg/mL puromycin for H42A and RJ206, respectively. Once the overexpression was assessed by Western blot, clonal selection was performed by diluting cells suspensions. For each cell line, three clones with higher NQO1 overexpression evaluated by Western blot analysis were pooled together. Three clones were also pooled for the mock cell lines.
10
Lentiviral Knockdown of PARG in Pancreatic Cancer
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