shRNA-mediated knockdown of SLC6A14 and β-catenin was carried out using a Lentivirus-based transduction system. A set of shRNA-lentiviral vectors for SLC6A14 was purchased from Open Biosystems (RHS4533-NM_007231); shRNA-lentiviral vectors for β-catenin were obtained from Addgene (shRNA1, #42543; shRNA2, #43544; pLKO.1, #84530). Lentiviral particles were generated in HEK293FT cell line by transfecting the plasmids along with the packaging plasmids pLP-1, pLP-2, and pVSVG (Invitrogen). Lipofectamine-2000 was used as the transfection reagent. After 72 h of transfection, the lentiviral supernatant was harvested and filtered through 0.45 µm filter. LS174T cells were infected with lentiviral particles carrying shRNA or empty vector for 24 h in a media containing 8 µg/ml Polybrene (Hexadimethrine bromide; Sigma) and cultured for an additional 24 h. Positive cells for transfection were selected by resistance to treatment with 3 µg/ml puromycin for 72 h. The resistant cells were maintained under the selective pressure of 0.5 µg/ml puromycin. Expression levels of mRNA and protein for SLC6A14 and β-catenin were measured by RT-qPCR and Western blot.
Shrna2
Manufactured by Addgene
ShRNA2 is a short hairpin RNA product designed for gene knockdown experiments. It provides a reliable and effective method for silencing target gene expression.
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Lab products found in correlation
2 protocols using shrna2
1
Silencing SLC6A14 and β-catenin in LS174T Cells
2
Generation of Fip200 Knockout and Rheb Overexpression in Tsc1 Cells
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Single guide RNA targeting mouse Fip200 5′-TTCTCTAGAAATAACACTAA-3′ was subcloned into pSpCas9(BB)-2A-GFP (PX458) (Addgene #48138) CRISPR-knockout (KO) vector according to the protocol previously reported.30 (link) Rictor shRNA-5 (TRCN0000123395) and shRNA-6 (TRCN0000123396) were obtained from Sigma-Aldrich MISSION® shRNA (short hairpin RNA). Non-target shRNA (#1864), raptor shRNA-1 (#21339) and shRNA-2 (#21340) were purchased from Addgene.
Fip200 KO in Tsc1iΔEC cells was generated by transiently transfecting PX458-sgFip200 into Tsc1iΔEC with Lipofectamine 3000 (Life Science Technology) according to the reagent protocol. Two days after transfection, single cells were seeded into 96-well plate by limited dilution method. Fip200 KO efficiencies in single clone expanded cells were confirmed by western blotting. pHAGE-CMV-Rheb (S16H)-IRES-eGFP-W (plasmid #32520) was purchased from Addgene. Lentiviral production containing Rheb (S16H) was transduced into LN229, and then EGFP (enhanced green fluorescent protein)-positive cells were sorted with flow cytometry and expanded.
All lentiviral productions were generated by transfecting into HEK293T cells with psPax2, pMD2.G and lentiviral plasmids, as described previously.31 (link)Tsc1iΔEC cells were then transduced by different lentivirus containing specific shRNA, followed by puromycin selection to generate stable knockdown tumour cells.
Fip200 KO in Tsc1iΔEC cells was generated by transiently transfecting PX458-sgFip200 into Tsc1iΔEC with Lipofectamine 3000 (Life Science Technology) according to the reagent protocol. Two days after transfection, single cells were seeded into 96-well plate by limited dilution method. Fip200 KO efficiencies in single clone expanded cells were confirmed by western blotting. pHAGE-CMV-Rheb (S16H)-IRES-eGFP-W (plasmid #32520) was purchased from Addgene. Lentiviral production containing Rheb (S16H) was transduced into LN229, and then EGFP (enhanced green fluorescent protein)-positive cells were sorted with flow cytometry and expanded.
All lentiviral productions were generated by transfecting into HEK293T cells with psPax2, pMD2.G and lentiviral plasmids, as described previously.31 (link)Tsc1iΔEC cells were then transduced by different lentivirus containing specific shRNA, followed by puromycin selection to generate stable knockdown tumour cells.
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