Plasmids suitable for Sleeping Beauty transfection and protein expression in an inducible manner were prepared based on the pSBtet-GP backbone (plasmid #60495; Addgene), digested with NcoI and HindIII-HF. The MYBL2 sequence was obtained by PCR amplification of a human cDNA clone (BioCat GmbH, Heidelberg, MYBL2 sequence [Gene ID: 4605] in pCMV-SPORT6 vector), whereas a non-coding sequence for the empty vector control was obtained by PCR amplification of the MCS of a pSBbi-GH vector (plasmid #60514; Addgene). NEBuilder HiFi DNA Assembly Master Mix (NEB) was used to gain the final transfection plasmids pSBtet_MYBL2 and pSBtet_LV, respectively.
Reporter gene constructs carrying a firefly luciferase (FLuc) sequence were prepared using a modified pSBtet-GP backbone. For the 5-LO promoter constructs, the tetracycline-on system was removed by PCR amplification of the two remaining backbone parts. In addition, the FLuc sequence of the previously described plasmid pN10 was amplified by PCR. PCR products were assembled using the NEBuilder HiFi DNA Assembly Master Mix to gain the promoterless vector pSBGP-LUC. ALOX5 promoter sequences N10 (−843 relative to ATG) and N6 (−2,530 relative to ATG) were obtained by PCR amplification of the previously described plasmids pN10 and pN0 (Sorg et al, 2006 (link)), respectively. The pSBGP_LUC backbone was linearized by PCR, and the final plasmids (pSBGP_pN10LUC; pSBGP_pN6LUC) were obtained using the NEBuilder HiFi DNA Assembly Master Mix. To generate the pSBGP_pN6ΔMYBLUC plasmid, deletion of an MYB binding site within the pN6 promoter region was necessary. Deletion was achieved by restriction digestion of the pSBGP_pN6LUC vector using EcoRV-HF and PvuI-HF (NEB) and PCR amplification of both the upstream and downstream regions of the binding site. Assembly using NEBuilder HiFi DNA Assembly Master Mix yielded the final pSBGP_pN6ΔMYBLUC plasmid. All plasmid constructs were verified by DNA sequencing.
To generate stable cell lines, the Sleeping Beauty (SB) co-transfection system was used. For each construct, HT-29 (0.5–0.7 × 106/3 ml) or HCT-116 (0.5–0.65 × 106/3 ml) cells were seeded into six-well microplates. After 24 h, the medium was changed to 2 ml Opti-MEM per well. Then, stable transfection was performed for 24 h using the Lipofectamine LTX Reagent according to the manufacturer’s protocol (LTX/DNA ratio: HT-29, 1:5; HCT-116, 1:3). All DNA mixes contained the SB100X transposase plasmid (kindly provided by Zoltan Ivics, Paul-Ehrlich-Institut) in a ratio of 1:20. After this, the medium was changed to the respective CGM. After another 24 h, puromycin (3 μg/ml) selection of the stably transfected cells was initiated. During the 9 d of selection, the medium was replaced every other day.
Reporter gene constructs carrying a firefly luciferase (FLuc) sequence were prepared using a modified pSBtet-GP backbone. For the 5-LO promoter constructs, the tetracycline-on system was removed by PCR amplification of the two remaining backbone parts. In addition, the FLuc sequence of the previously described plasmid pN10 was amplified by PCR. PCR products were assembled using the NEBuilder HiFi DNA Assembly Master Mix to gain the promoterless vector pSBGP-LUC. ALOX5 promoter sequences N10 (−843 relative to ATG) and N6 (−2,530 relative to ATG) were obtained by PCR amplification of the previously described plasmids pN10 and pN0 (Sorg et al, 2006 (link)), respectively. The pSBGP_LUC backbone was linearized by PCR, and the final plasmids (pSBGP_pN10LUC; pSBGP_pN6LUC) were obtained using the NEBuilder HiFi DNA Assembly Master Mix. To generate the pSBGP_pN6ΔMYBLUC plasmid, deletion of an MYB binding site within the pN6 promoter region was necessary. Deletion was achieved by restriction digestion of the pSBGP_pN6LUC vector using EcoRV-HF and PvuI-HF (NEB) and PCR amplification of both the upstream and downstream regions of the binding site. Assembly using NEBuilder HiFi DNA Assembly Master Mix yielded the final pSBGP_pN6ΔMYBLUC plasmid. All plasmid constructs were verified by DNA sequencing.
To generate stable cell lines, the Sleeping Beauty (SB) co-transfection system was used. For each construct, HT-29 (0.5–0.7 × 106/3 ml) or HCT-116 (0.5–0.65 × 106/3 ml) cells were seeded into six-well microplates. After 24 h, the medium was changed to 2 ml Opti-MEM per well. Then, stable transfection was performed for 24 h using the Lipofectamine LTX Reagent according to the manufacturer’s protocol (LTX/DNA ratio: HT-29, 1:5; HCT-116, 1:3). All DNA mixes contained the SB100X transposase plasmid (kindly provided by Zoltan Ivics, Paul-Ehrlich-Institut) in a ratio of 1:20. After this, the medium was changed to the respective CGM. After another 24 h, puromycin (3 μg/ml) selection of the stably transfected cells was initiated. During the 9 d of selection, the medium was replaced every other day.
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