For knockdown of the α2δ1 subunit, siRNA target sequences corresponding to the α2δ1 coding region (CACNA2D1, GenBank accession number NM_009784.2 ) (Obermair et al., 2005 (link)) were selected and tested for efficient knockdown. The siRNA was expressed as shRNA under the control of a U6 promoter (derived from the pSilencer1.0-U6 siRNA expression vector, Ambion) cloned into the pβA-eGFP plasmid (Obermair et al., 2010 (link)). For lentiviral expression, α2δ1 shRNA was cloned into pHR as previously described (Subramanyam et al., 2009 (link)). For knockdown of the α2δ3 subunit, four 29mer shRNA constructs against rat Cacna2d3 (Gene ID 306243) cloned in lentiviral GFP vector (pGFP-C-shLenti Vector, catalog #TR30023) were ordered from OriGene Technologies (catalog #TL713428). Based on their specificity for rat and mouse α2δ3, two of these constructs were tested for their knockdown efficiency where the construct “C” was evaluated to results in a reduction of α2δ3 expression down to 40%-50%. As control for α2δ-knockdown experiments, a noneffective 29-mer scrambled shRNA cassette cloned into pGFP-C-shLenti Vector (catalog #TR30021; OriGene Technologies) was used.
Pgfp c shlenti vector
Manufactured by OriGene
Sourced in United States
The PGFP-C-shLenti vector is a lentiviral expression vector designed for the expression of short hairpin RNA (shRNA) in mammalian cells. It contains a puromycin resistance gene for selection of transduced cells and a green fluorescent protein (GFP) reporter for monitoring transduction efficiency.
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Lab products found in correlation
Pmd2 g, by Addgene (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (4 mentions)
Pspax2, by Addgene (3 mentions)
Pgfp c shlenti, by OriGene (2 mentions)
Lipofectamine 3000 transfection reagent, by Addgene (2 mentions)
Peg it virus precipitation solution, by System Biosciences (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Mir 298, by Genechem (2 mentions)
Mir ctrl, by Genechem (2 mentions)
Anti mir ctrl, by Genechem (2 mentions)
Anti mir 298, by Genechem (2 mentions)
Pcdna3, by Santa Cruz Biotechnology (2 mentions)
Psilencer1.0 u6 sirna expression vector, by Thermo Fisher Scientific (1 mentions)
Turbofectin transfection reagent, by OriGene (1 mentions)
Cox 2 silencer select sirna, by Thermo Fisher Scientific (1 mentions)
Mission lentiviral packing mix, by Merck Group (1 mentions)
Hek293ft, by Thermo Fisher Scientific (1 mentions)
Genez orf database, by GenScript (1 mentions)
Pspax, by Addgene (1 mentions)
35 protocols using pgfp c shlenti vector
1
Knockdown of α2δ1 and α2δ3 Subunits
2
Knockdown of ANGPTL4 in MDA-MB-231 Cells
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293T cells grown to 70% confluence in DMEM were transfected with pGFP-C-shLenti vectors expressing four unique 29-mer shRNAs for human ANGPTL4 (ANGPTL4 human shRNA, OriGene Technologies, Inc.), and a control 29-mer scrambled shRNA cassette (Control shRNA) in the pGFP-C-shLenti Vector (OriGene Technologies, Inc.) by using TurboFectin transfection reagent (OriGene Technologies, Inc.) following the manufacturer’s instructions. The culture media was changed after 12–18 h of incubation. Two batches of the viral supernatants of 293T cells were collected 48 and 72 h after transfection and combined and then filtered to remove cellular debris. MDA-MB-231 cells with stable knockdown of ANGPTL4 (Angptl4shRNA) and an empty vector control (ConshRNA) were established by incubation of MDA-MB-231 cells with the viral supernatant for 5 h and puromycin selection for at least 2 weeks. The transfection efficiency was monitored by positive GFP expression. Efficient shRNA-mediated knockdown of ANGPTL4 in MDA-MB-231 cells was confirmed by qPCR. Those colonies with the best knockdown efficiency of ANGPTL4 were used for experiments.
3
Modulating Key Cellular Regulators
4
SHMT2 Silencing in Cancer Cells
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SHMT2 expression was silenced using a commercial pGFP-C-shLenti vector (catalog No: TL309453V, OriGene Technologies, Inc., Rockville, MD, USA). This vector was designed to specifically inhibit SHMT2 expression (shSHMT2-1:5′-AAG ACTGCCAAGCTCCAGGATTTCAAATC-3′; shSHMT2-2:5′- AAGTCAAAGCACACCTGCTGGCAGACATG-3′) and encodes green fluorescent protein as a marker of transfection. Lenti-shRNA scramble control particles were used as a negative control (catalog No: TR30021V, OriGene Technologies, Inc., Rockville, MD, USA). FADU and SNU1041 cells were infected with shRNA-expressing lentiviruses and selected with puromycin.
5
Lentivirus Transduction of Primary Hippocampal Neurons
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Some cultures were infected with lentiviruses five days before experiments. Lentiviruses were generated by transfecting HEK293T with polyethyleneimine and a mixture of three plasmids: envelope vector pMD2.G, packaging vector psPAX2 (AddGene #12259 and #12260 were gifts from Didier Trono), pGFP-C-shLenti vector (OriGene, Rockville, MD, USA, TR30021, referred to as mock shRNA), shRNA STIM1 or shRNA STIM2 vectors (a kind gift from prof. I. Bezprozvanny, Southwestern. Medical Center, Dallas, TX, USA). The supernatant with viruses was harvested 48 and 72 h after transfection, filtered, and concentrated by centrifugation (47,000 g, 2 h, 4 °C). Pellets were resuspended in NeuroBasal-A, aliquoted, and stored at −80 °C. Transduction efficacy was confirmed by western blotting and immunostaining of primary hippocampal neuron cultures of C3Ha mice.
6
Lentiviral shRNA Knockdown of PHLPP-1 in Rats
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The lentiviral plasmids encoding shRNAs for PHLPP-1 were constructed in pGFP-C-shLenti vector (Catalog # TR30023) purchased from OriGene Technologies, Inc. A plasmid carrying a non-targeting sequence was used to create the control cells. For virus packaging, the control or PHLPP-specific shRNA constructs were co-transfected with Mission lentiviral packing mix (Sigma-Aldrich) [39 (link)]. Sprague–Dawley rats underwent gene transfer via aortic cross-clamping as previously described [25 (link)]. After dissection of the aorta and pulmonary artery, lentiviral shPHLPP-1 was injected into the left ventricular cavity through a 22G catheter while the aorta and pulmonary artery were cross-clamped for 50 s. In negative control animals, sh-Con lentivirus was injected into the left ventricular cavity while the aorta and pulmonary artery were cross-clamped for 50 s. After cross-clamping was released, the chest was closed. Myocardial PHLPP-1 expression was analyzed 72 hours later by western blotting.
7
Lentiviral Transduction of ErbB and PDGFRα Genes
8
Lentiviral Mfn2 Knockdown in Neurons
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To induce Mfn2 knockdown, commercially available 29mer short-hairpin RNA (shRNA) constructs in lentiviral GFP vector (Origene, Rockville, USA, TL712567) were used. Two constructs, sh-Mfn2 B and sh-Mfn2 D, were selected for the experiments, based on satisfactory efficiency in Mfn2 reduction with a relatively low toxicity. As a negative control, a non-effective 29-mer scrambled shRNA cassette in pGFP-C-shLenti Vector was used (scrRNA; Origene). Viral production was performed as described in Gruszczynska-Biegala et al. (2020) [34 ]. Neurons were infected with sh-Mfn2 and scrRNA- carrying lentiviruses on DIV4 with a viral infection efficiency exceeding 90%. The experiments were performed 6 days post transduction (DIV 10).
9
Lentiviral Knockdown of ASPP2κ in CRC
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Recombinant lentiviral particles expressing a custom-made 29-mer short hairpin (sh) RNA against ASPP2κ were produced and transduced into the CRC cells according to the manufacturer’s protocols. Empty vector (EV) strains served as controls.
Briefly, a pre-selected custom synthesized lentiviral construct was designed (pGFP-C-shLenti vector; Origene) containing a shRNA expression cassette against ASPP2κ, driven by an U6 promoter, a puromycin resistance marker, driven by a SV40 promoter and a tGFP, driven by a CMV promoter. HEK293T cells were lipofected (Lipofectamine 2000; Thermo Fisher) for lentiviral particle production following the manufacturer’s protocol (packaging mix, Dharmacon). The lentiviral particles were used to transduce HCT116 or DLD-1 cells to induce stable hairpin expression against ASPP2κ. The transduction efficiency was evaluated determining GFP expression, while puromycin was used as a selection marker.
Briefly, a pre-selected custom synthesized lentiviral construct was designed (pGFP-C-shLenti vector; Origene) containing a shRNA expression cassette against ASPP2κ, driven by an U6 promoter, a puromycin resistance marker, driven by a SV40 promoter and a tGFP, driven by a CMV promoter. HEK293T cells were lipofected (Lipofectamine 2000; Thermo Fisher) for lentiviral particle production following the manufacturer’s protocol (packaging mix, Dharmacon). The lentiviral particles were used to transduce HCT116 or DLD-1 cells to induce stable hairpin expression against ASPP2κ. The transduction efficiency was evaluated determining GFP expression, while puromycin was used as a selection marker.
10
Stable Cebpg Knockdown in 50B11 Cells
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Stable knockdown of Cebpg in 50B11 cells was done by transfecting 50B11 cells with pGFP-C-shLenti carrying shRNA against Cebpg (1μg/ml; TL709448; Origene, Rockville, MD) following the manufacturer’s recommendations. A 29-mer scrambled shRNA cassette in the pGFP-C-shLenti vector (TR30021; Origene) was used as the off-target control. Transduced cells then underwent puromycin selection and visible confirmation for GFP. Cebpg knockdown efficiency compared to control was verified by Western blot (Online Resource 3).
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