PMC:1702488 / 4700-6914
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1702488","sourcedb":"PMC","sourceid":"1702488","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1702488","text":"Cell culture and reporter gene constructs\nNS20Y and HeLa cells were routinely grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2. PC12 cells were cultured in 10% CO2 in DMEM with 10% donor horse serum and 5% FBS (7).\nThe pGL4.7 (−4744 to +1, the translation start site was designated as +1) was generated by ligation of the PCR product (−249 to +1) with the BamHI and NcoI digested pL4.7K (−4744 to −249) (19). PCR was performed using genomic DNA from mouse NS20Y cells as a template and an upstream sense oligonucleotide (5′-GCCTCTGGATCCCTCACAGCCCAT-3′), containing a BamHI site, and a downstream antisense oligonucleotide (5′-GGCGCTGCTGTCCATGGTTCTGAA-3′) containing a NcoI site. The pGL4.7NRSP, pGL4.7preNRmSP, pGL4.7NRSPm and pGL4.7preNRmSPm constructs were generated by ligation of the pGL4.7 DNA digested with NcoI and the double-strand oligomers (pGL4.7NRSP; wild type of NRSE and Sp-binding sequence, pGL4.7NRmSP; mutated NRSE and wild type of Sp-binding sequence, pGL4.7NRSPm; wild type of NRSE and mutated Sp-binding sequence, pGL4.7NRmSPm; mutated NRSE and mutated Sp-binding sequence) containing NcoI site at both 5′ and 3′ ends (for pGL4.7NRSP: 5′-TTCAGAACCATGGACAGCAGCGCGCCGGCCCATGGATTCTTC-3′; for pGL4.7preNRmSP: 5′-TTCAGA ACCATGGA ATAGTTGCGCGCCGGCCCATGGATTCTTC-3′; for pGL4.7NRSPm: 5′-TTCAGAACCATGGACAGCAGCGCATATGCCCATGGATTCTTC -3′; for pGL4.7preNRmSPm: 5′-TTCAGAACCATGGAATAGTTGCGCATATGCCCATGGATTCTTC-3′) (The underlines indicate mutated nucleotides for NRSE and Sp3 core binding sites).\nThe pGL4.7NRmSP and pGL4.7NRmSPm constructs were finally generated by PCR site-directed mutagenesis using high-fidelity Pfu DNA polymerase according to the manufacturer's protocol (Quikchange TM; Stratagene). In vitro mutagenesis was carried out on MOR promoter linked to luciferase gene reporter (pGL4.7preNRmSP and pGL4.7preNRmSPm) using primers as follows: for pGL4.7NRmSP: 5′-TTCAGAACCATAAAATAGTTGCGCGCCGGCCCATGGATTCTTC-3′; 5′-GAAGAATCCATGGGCCGGCGCGCAACTATTTTATGGTTCTGAA-3′; for pGL4.7NRmSPm: 5′-TTCAGAACCATAAAATAGTTGCGCATATGCCCATGGATTCTTC-3′; 5′-GAAGAATCCATGGGCATATGCGCAACTATTTTATGGTTCTGAA-3′). The mutated nucleotides are underlined.","divisions":[{"label":"Title","span":{"begin":0,"end":41}}],"tracks":[{"project":"2_test","denotations":[{"id":"17130167-12446692-76660561","span":{"begin":285,"end":286},"obj":"12446692"},{"id":"17130167-9177274-76660562","span":{"begin":478,"end":480},"obj":"9177274"}],"attributes":[{"subj":"17130167-12446692-76660561","pred":"source","obj":"2_test"},{"subj":"17130167-9177274-76660562","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#c6ec93","default":true}]}]}}