To construct stable COX-2-Overexpression cell lines, mouse Cox2 cDNA was obtained from Ptgs2 (NM_011198) Mouse Tagged ORF Clone (Clone ID MR227684, OriGene). The cDNA was cloned into the plasmid pLVX-IRES-ZsGreen1 to create the pLVX-COX-2-IRES-ZsGreen1 vector. Empty pLVX-IRES-ZsGreen1 was used as negative control. Recombinant construct with three helper plasmid pLP1, pLP2 and VSVG were transiently transfected into 293T cells using Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. After 48 h, the supernatant was collected and lentivirus particles were concentrated with 4 M NaCl and PEG6000. Then fresh virus was used to infect RAW264.7 cells with 10 ng/µl polybrene. GFP positive polyclone cells were sorted by a flow cytometer to generate stable cell lines. The polyclone cells were measured by western blotting.
To establish stable COX-2-Knockdown cell lines, two shRNA sequences were chosen for targeting COX-2 by NCBI BLAST (Tiedemann et al., 2012 (link)). The double-stranded oligonucleotides were annealed and inserted into plasmid pLL3.7 lentivirus vectors. The transfection and cell line sorting procedures were same as the ones used for construction of overexpression cell lines. Primer sequences in this study were listed inSupplementary Table 1 .
To establish stable COX-2-Knockdown cell lines, two shRNA sequences were chosen for targeting COX-2 by NCBI BLAST (Tiedemann et al., 2012 (link)). The double-stranded oligonucleotides were annealed and inserted into plasmid pLL3.7 lentivirus vectors. The transfection and cell line sorting procedures were same as the ones used for construction of overexpression cell lines. Primer sequences in this study were listed in
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