MGL knockdown was achieved by the lentivirus-mediated small hairpin RNA (shRNA) expression as we previously described6 (link). Three different nucleotide sequences targeting the human MGL were as follows: shRNA1 5′-ccaggacaagactctcaagat-3′; shRNA2 5′-caactccgtcttccatgaaat-3′; and shRNA3 5′-ccaatcctgaatctgcaacaa-3′. The scramble shRNA construct was commercially obtained from Addgene, Inc. (Cambridge, MA). Lentivirus preparation, expansion and infection were performed per protocol provided by Addgene.
Shrna1
Manufactured by Addgene
ShRNA1 is a short hairpin RNA (shRNA) product. It is a tool used to induce RNA interference (RNAi) and knockdown the expression of target genes in cells.
Automatically generated - may contain errors
2 protocols using shrna1
1
Lentiviral-mediated MGL knockdown
2
Silencing SLC6A14 and β-catenin in LS174T Cells
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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.
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