Transient EpCAM knockdown was achieved by siRNA transfection (50 nM ) for 72 h using lipofectamine transfection reagent (Invitrogen, Carlsbad, CA, USA). Sequences: EpCAM siRNA, 5′-UGCCAGUGUACUUCAGUUG-3′ control siRNA, 5′-UCGUCCGUAUCAUUUCAAU-3′. Stable EpCAM knockdown was achieved by stable shRNA expression introduced by lentiviral pGIPZ vectors (Thermo Scientific, Rockford, IL, USA) followed by puromycine selection as previously described (Ploner et al, 2008 (link); Martowicz et al, 2012 (link)). For miRNA transfection, hsa-miR-200c, hsa-miR-205, and miRNA mimic negative control were purchased from Dharmacon (Chicago, IL, USA). Cells were transfected twice during a period of 6 days with 25 nM miRNAs using lipofectamine transfection reagent (Invitrogen).
Lentiviral pgipz vectors
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lentiviral pGIPZ vectors are gene delivery tools used to introduce genetic material into target cells. These vectors are lentiviral-based, allowing for efficient transduction and stable integration of the desired genetic sequences into the host cell genome.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine transfection reagent, by Thermo Fisher Scientific (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 rnaimax, by Thermo Fisher Scientific (1 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
4 protocols using lentiviral pgipz vectors
1
EpCAM Knockdown Using siRNA and shRNA
2
Silencing SMAD and ALK-5 in Glioma Cells
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To silence the expression of SMAD2, SMAD3, SMAD4, or ALK-5, GIC were transiently transfected by electroporation (Neon transfection system, Invitrogen) using siRNA pools (ON-TARGET plus human SMAD2 siRNA-SMART pool L-003561-00, ON-TARGET plus human SMAD3 siRNA-SMART pool L-020067-00-0020, ON-TARGET plus human SMAD4 siRNA- SMART pool L-003902-00 and ON-TARGET plus human ALK-5 siRNA- SMART pool L-003929-00 (Dharmacon, Lafayette, CO). Non-targeting siRNA pool was used as a negative control. Lentiviral pGIPZ vectors encoding c-MET-specific (Oligo ID V3LHS_642486) or non-silencing control shRNA (Oligo ID RHS4346) were purchased from Thermo Scientific (Waltham, MA). Glioma cells were transduced with lentiviral particles produced as described37 (link). Stably transduced clones were isolated with 4 µg/ml puromycin and subjected to analyses and assays after 1–5 passage post selection.
3
Silencing DOCK9 and CDC42 in MDA-MB-231 Cells
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MDA-MB-231/Brain cells (1.5 ×105 per well) were seeded onto 6-well plates (Corning) and transfected with 10 nM siRNA using Lipofectamine 2000 RNAiMax (Invitrogen) and Opti-MEM I Reduced Serum Medium (Gibco) according to the manufacturer’s recommendations. Assays were performed 48 h after transfection. For stable knockdown MDA-MB-231/Brain cells expressing shRNAs and EGFP, or empty vector EGFP control were generated by lentiviral infection in the presence of polybrene (8 μg ml−1), and EGFP expressing cells were FACS sorted and expanded for use in assays. Lentiviral vectors (pGIPZ) were obtained from Open Biosystems. The sequences of DOCK9 and CDC42 siRNAs, and DOCK4 shRNAs were as previously listed25 (link).
4
Stable Knockdown of HUVEC using Lentivirus
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For stable knockdown HUVEC stably expressing shRNAs or empty vector EGFP control were generated by lentiviral infection in the presence of polybrene (8 μg ml−1). Lentiviral vectors (pGIPZ) harbouring shRNAs and EGFP were obtained from Open Biosystems. Virus production was according to http://tronolab.epfl.ch/webdav/site/tronolab/shared/protocols/LVproduction.pdf and supernatants for infection were used at 1:4 dilution of harvested virus-containing supernatants. HUVEC were infected at ∼70% confluence and used in biochemical assays 48 h after infection, or seeded onto coculture assays following fluorescence-activated cell sorting of EGFP-expressing cells. Ecotropic retrovirus producer lines used in the xenograft co-injection model were generated by transfection of TE-FLY-Mo packaging cells15 (link) by calcium phosphate of retroviral vectors harbouring shRNAs, followed by selection in 0.5 μg ml−1 puromycin. High-titre producer clones were chosen following titration of harvested retroviruses in 3T3 fibroblasts following infection and selection with 0.5 μg ml−1 puromycin15 (link).
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