293T were cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 5% FBS containing Antibiotic/Antimycotic. Lentiviral shRNA (OpenBiosystems, Lafayette, CO) particles were generated according to Thermoscientific specifications and as described previously (Chudnovsky et al., 2005 (link), Hansen and Johansen, 2011 (link), Lazarov et al., 2002 (link), Ridky et al., 2010 (link)) . For the production of viral particles, lentiviral constructs were co-transfected with viral packaging plasmids pCMVΔR8.91 and pUC-MDG into 293T cells using Fugene 6 Transfection Reagent (Promega, Fitchburg, WI).
Lentiviral shrna
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lentiviral shRNA is a laboratory tool used to deliver short hairpin RNA (shRNA) into cells. shRNA is designed to target and silence specific genes, enabling the study of gene function. The lentiviral vector provides an efficient method for introducing the shRNA into a wide range of cell types, including both dividing and non-dividing cells.
Automatically generated - may contain errors
Lab products found in correlation
Fugene 6 transfection reagent, by Promega (2 mentions)
Tt2609 c02, by Leibniz Institute DSMZ (2 mentions)
Ftc 133, by Merck Group (1 mentions)
Pcmv vsv g, by Addgene (1 mentions)
Pspax2, by Addgene (1 mentions)
Dharmafect 1 reagent, by Thermo Fisher Scientific (1 mentions)
Cxcr4, by Merck Group (1 mentions)
Lipofectamine ltx plus reagent, by Thermo Fisher Scientific (1 mentions)
Cxcr4, by Thermo Fisher Scientific (1 mentions)
Cxcr7, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Arrest in reagent, by Thermo Fisher Scientific (1 mentions)
6 protocols using lentiviral shrna
1
Lentiviral shRNA Particle Production
2
Lentiviral and Retroviral Transduction
3
Lentiviral Transduction of FTC Cell Lines
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Human FTC cell line TT2609-C02 was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ no.: ACC 510). The cell line originated from local tumour recurrence of a secondary RAI refractory FTC. Under culture conditions the cell line demonstrates no relevant 125I uptake45 (link).
The human FTC cell line FTC133 was purchased from Sigma-Aldrich (catalogue no.: 94060901). The cell line was obtained from a patient with metastatic FTC and does not demonstrate iodine uptake under culture conditions46 (link), 47 . Authenticity of both cell lines was confirmed by Short Tandem Repeat (STR) DNA profiling.
For lentiviral transduction 2.5 × 106 HEK293FT cells were incubated in DMEM/10% FCS over night at 37 °C and 5% CO2. Transfection was performed using Lipofectamine2000TM with envelope plasmid pCMV-VSVG, packaging plasmid psPAX2 (both Addgene, Cambridge, MA, USA) and respective targeting vectors for XIAP (clone V2LHS-94578; mature antisense sequence: TTACAAGTGACTAGATGTC), survivin (clone V2LHS_262484; mature antisense sequence: TTCCTAAGACATTGCTAAG) or GIPZ non-silencing Lentiviral shRNA (all Open Biosystems, Dharmacon, Lafayette, CO, USA).
Lentiviral supernatant was harvested and FTC cell lines transduced using polybrene. Knockdown was validated by western blot analysis.
The human FTC cell line FTC133 was purchased from Sigma-Aldrich (catalogue no.: 94060901). The cell line was obtained from a patient with metastatic FTC and does not demonstrate iodine uptake under culture conditions46 (link), 47 . Authenticity of both cell lines was confirmed by Short Tandem Repeat (STR) DNA profiling.
For lentiviral transduction 2.5 × 106 HEK293FT cells were incubated in DMEM/10% FCS over night at 37 °C and 5% CO2. Transfection was performed using Lipofectamine2000TM with envelope plasmid pCMV-VSVG, packaging plasmid psPAX2 (both Addgene, Cambridge, MA, USA) and respective targeting vectors for XIAP (clone V2LHS-94578; mature antisense sequence: TTACAAGTGACTAGATGTC), survivin (clone V2LHS_262484; mature antisense sequence: TTCCTAAGACATTGCTAAG) or GIPZ non-silencing Lentiviral shRNA (all Open Biosystems, Dharmacon, Lafayette, CO, USA).
Lentiviral supernatant was harvested and FTC cell lines transduced using polybrene. Knockdown was validated by western blot analysis.
4
ROBO1 Silencing via Lentiviral shRNA
5
Characterization of Rho GTPase Signaling in Breast Cancer and Cell Lines
6
Efficient ALDH1A1 Knockdown in A2780/CP70 Cells
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Six different pGIPZ Lentiviral shRNA glycerol stocks against ALDH1A1 and one negative control shRNA (Thermo Scientific) were transfected according to the manufacturer’s instructions. A2780/CP70 cells were plated at a density of 2×105 cells per well of a 6-well plate for 18–24 hours. For each well, 2 µg shRNA plasmid DNA (pGIPZ) were transfected into A2780/CP70 using Arrest-In reagent (Thermo Scientific, USA). Puromycin (0.8 µg/ml) was used to select the transfected cells. After optimization, ALDH1A1-knockdown efficiencies of individual shRNAs were evaluated using real-time quantitative PCR and Western Blot. Vector 398453 demonstrated the most efficient transfection with over 95% knockdown efficacy of ALDH1A1 and was used for all shRNA knockdown experiments.
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