Pgl3 basic firefly luciferase reporter plasmid

The wide-type and mutant FOXM1 3' UTR reporter plasmid was constructed by cloning PCR-amplified sequences from the 3' UTR of FOXM1 cDNA into a pGL3-basic firefly luciferase reporter plasmid (Promega, Madison, WI, USA). CAOV3 cells transduced with HuR-expressing vector were seeded into 24-well plates and transfected with 400 ng of FOXM1 3' UTR reporter plasmid (WT or Mutant). To normalize the transfection efficiency, cells were cotransfected with 50 ng of pRL-TK containing renilla luciferase. After 48 h, cells were washed with PBS and lysed using passive lysis buffer. Luciferase activity was assessed using a Dual-Luciferase Reporter Assay system (Promega) following the manufacturer’s instructions.

A genomic region including 1680 bp of sequence upstream of the first exon of cers2b was cloned into the TOPO TA vector and transferred to the Kpn1/Xho1 sites of the pGL3 basic firefly luciferase reporter plasmid (Promega). To generate shorter promoter constructs the same 3’ primer was used along with 5’ primers targeting progressively proximal sequences. These PCR fragments were cloned into TOPO TA and transferred to the Kpn1/Xho1 sites of the pGL3 basic firefly luciferase reporter plasmid. A construct containing the first 400 bp of sequence (the most distal 5’ sequences of the full-length promoter construct) was also generated by PCR and cloned into a pGL3 promoter firefly luciferase reporter plasmid containing an SV40 promoter (Promega). Primer sequences are:
1680 bp: 5’-ATGAAAATATATGTGAATAAACTGCAA
1631 bp: 5’-AAACGAAGGTTGAGGGAACG
1621 bp: 5’-TGAGGGAACGTTTCTTCATG
1611 bp: 5’-TTTCTTCATGGTTTAAAAGCGTTC
1597 bp: 5’-AAAAGCGTTCTTTAGATTATTAAAATG
1498 bp: 5’-AAAACTGAATATTGAGTAGGGGG
1281 bp: 5’-AAACTCAGTCTCCCGCAAAGTT
881 bp: 5’-TAGGCTATGATGTTGGGTTACTTTGT
common reverse primer: 5’-GTAACTCACGTAGGATCGGC
Two point mutations in the HOX domain (R121W, R122S) were generated using the full-length 1680 bp pGL3 construct via site directed mutagenesis. Likewise, two point mutations in the Lag domain (H212D, H213D) were similarly generated. Mutations were confirmed by directly sequencing the plasmids.

The human and mouse α-klotho gene promoter sequence was amplified with PCR using genomic DNA extracted from the mouse tail or BAC clone (RP11-720E2) containing the entire human α-klotho gene as templates. Deletion mutants were prepared with standard PCR procedures. Promoter sequences containing point mutations were generated using the Quick Change Site-directed Mutagenesis kit (Agilent Technologies). The promoter fragments were inserted into PGL3 basic firefly luciferase reporter plasmid (Promega). All inserted DNAs were sequenced and verified. Transfection of reporter plasmids was performed using the X-treamGENE9 DNA transfection reagent (Roche) according to the manufacturer's instructions. The luciferase assays were performed using the Luciferase assay systems kit (Promega). Cytomegalovirus promoter-renilla luciferase plasmid or SV40 promoter-β-galactosidase plasmid was used as an internal control.