NK1R knockdown was performed as previously described [23 (link)]. The lentivirus particles containing shNK1R were obtained from OBIO Technology (Shanghai, China). The shRNA sequences were cloned into lentiviral vector pLKD-CMV-EGFP-Puro respectively (shctrl: 5´-TTCTCCGAACGTGTCACGT-3´; shNK1R 1#: 5´-GCAACCAGCCTG GCAAATT-3´; shNK1R2#: 5´-GCCTGTTCTACTGCAAGTT-3´). Cells were transfected with the lentivirus particles according to the manufacturer’s instructions followed by puromycin selection (5 μg/ml, Solarbio, Beijing, China). For NK1R overexpression, pLenti-EGFP-Puro-CMV-NK1R plasmid (OBIO) was transfected into cells with GC Liposomal Transfection Reagent (Genecarer, Xi’an, China) followed with puromycin selection.
A dual sgRNA CRISPR/Cas9 system was used to delete the ARE1 sequence in the enhancer region of the NK1R gene. Briefly, sgRNA sequences (sgRNA-F: 5’-GTACGA ATAGCCATCATATCCTGG -3’; sgRNA-R: 5’-GTCCTAAGAGCATTACACCTG AGG-3’) were cloned into the vector of Lenti-CRISPR-dual gRNA. The cells were transfected with Lenti-CRISPR-dual gRNA-ARE1 or vector control plasmid with GC Liposomal Transfection Reagent for 48 h, then harvested for the analysis of ARE1 deletion efficiency using Takara ExTaq PCR kit (Dalian, China) with the forward primer 5’-AGCGGTTTCCCAGTAGAGTC-3’ and the reverse primer 5’-AAGGGTTCAGCATGTTCTGC-3’.
A dual sgRNA CRISPR/Cas9 system was used to delete the ARE1 sequence in the enhancer region of the NK1R gene. Briefly, sgRNA sequences (sgRNA-F: 5’-GTACGA ATAGCCATCATATCCTGG -3’; sgRNA-R: 5’-GTCCTAAGAGCATTACACCTG AGG-3’) were cloned into the vector of Lenti-CRISPR-dual gRNA. The cells were transfected with Lenti-CRISPR-dual gRNA-ARE1 or vector control plasmid with GC Liposomal Transfection Reagent for 48 h, then harvested for the analysis of ARE1 deletion efficiency using Takara ExTaq PCR kit (Dalian, China) with the forward primer 5’-AGCGGTTTCCCAGTAGAGTC-3’ and the reverse primer 5’-AAGGGTTCAGCATGTTCTGC-3’.
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