PMC:5608918 / 30008-32016 JSONTXT

{"project":"2_test","denotations":[{"id":"28785074-12386374-79074356","span":{"begin":177,"end":179},"obj":"12386374"},{"id":"28785074-18781178-79074357","span":{"begin":439,"end":441},"obj":"18781178"},{"id":"28785074-21404460-79074358","span":{"begin":443,"end":445},"obj":"21404460"}],"text":"Preparation of pGL3-MDM2 promoter P1/P2\nLuciferase and β-galactosidase expression vectors under the control of the P1 or P2 MDM2 promoters were prepared as described previously.54 Cells were cultured normally, then their genomic DNA was extracted and analyzed for purity and concentration by agarose gel electrophoresis and spectrophotometry. The primers for promoters P1 and P2 were designed according to the reported human MDM2 sequence,55, 56 and XhoI and HindIII restriction sites were introduced upstream or downstream of the primers. For the P1 promoter, the upstream primer was 5′-CTGGGGAGTCTCGAGGGACC-3′ and the downstream primer was 5′-CTGCCTCAAGCTTCTTACGT-3′. For the P2 promoter, the upstream primer was 5′-CCTGTGTCTCGAGAAGATGG-3′ and the downstream primer was 5′-CTAAGCTTCGAGTCTCCTGT-3′. After amplification by PCR and purification by agarose electrophoresis, target DNA segments (P1 or P2) were recovered. Then the target DNA segments were linked with the pGEM-T vector. The ratio of the target DNA segments/vector was 3:1. The linkage reaction was performed at 16 °C overnight. The products of the linkage reaction were transferred into competent DH5α cells (Clontech, Mountain View, CA, USA). After the bacteria were cultured in LB agarose medium and their DNA were analyzed by cleavage electrophoresis, the positive clones were selected for amplification and preparation of a small amount of the target plasmid DNA. The plasmid DNA, pGEM-MDM2 promoter, was digested by XhoI and HindIII. The restriction fragments were analyzed by 1% agarose gel electrophoresis. The sequence of the restriction fragments was also measured by Invitrogen Co. to ensure that the gene sequence of the recombinant plasmid pGEM-MDM2 promoter was correct. Plasmid pGL3-enhancer was also digested by XhoI and HindIII and formed cohesive ends. Then, the fragment pGEM-MDM2, which included P1 or P2 was linked with the pGL3-enhancer linear fragment, and the respective products were called pGL3-MDM2 P1 or pGL3-MDM2 P2."}