Oligonucleotides used for the production of promoter fragments, plasmid construction, mobility shift assays, site-directed mutagenesis, RT-PCR, and quantitative PCR
Regarding the oligonucleotides used for the mobility shift assays and site-directed mutagenesis, the HNF1 recognition motif in the hOAT3 promoter region is underlined. Bold type indicates the difference in the sequence of the per and mut compared with wt sequence found in the hOAT3 promoter.
Oligonucleotide | Orientation | Sequence (5′ to 3′) | ||
|---|---|---|---|---|
| Primers and oligonucleotides used for cloning of 5′-flanking regions | ||||
| hOAT3 | ||||
| −1471 | Forward | CGGGGTACCGGAACAGAGAGGTAAAGGC | ||
| −644 | Forward | CGGGGTACCGAGAGAAGCCTGTCCATTG | ||
| −308 | Forward | CGGGGTACCGATTCCTTCCCAGAATCTCC | ||
| +6 | Reverse | CCCAAGCTTCAAGCTGTGTTTGTGCCTCC | ||
| −35/+6 | Sense | CCCTTATATAAGCCCCCCTGGGGGAGGCACAAACACAGCTTGA | ||
| +6/−35 | Antisense | AGCTTCAAGCTGTGTTTGTGCCTCCCCCAGGGGGGCTTATATAAGGGGTAC | ||
| mOat3 | ||||
| −156 | Forward | CGGGGTACCATCAACAGCCTTGGCTGAGG | ||
| +6 | Reverse | CCCAAGCTTCAAGCTGTTTGTGTCTCC | ||
| Primers used for cloning of HNF1α and HNF1β | ||||
| HNF1α | Forward | AAGCTTGCCATGGTTTCTAAACTGAGCC | ||
| Reverse | GAATTCTGGTTACTGGGAGGAAGAGGCC | |||
| HNF1β | Forward | AAGCTTGAAAATGGTGTCCAAGCTCACG | ||
| Reverse | GAATTCGGCATCACCAGGCTTGTAGAGG | |||
| Oligonucleotides used for construction of EMSA probe and competitor or site-directed mutagenesis | ||||
| wt | Sense | CGCAAAAGAAAGTCAAACATTAGCCCGGGAAACAGC | ||
| per | Sense | CGCAAAAGAAAGTTAATCATTAACCCGGGAAACAGC | ||
| mut | Sense | CGCAAAAGAAAGTCACACATCGCCCCGGGAAACAGC | ||
| Primers used for RT-PCR or quantitative PCR | ||||
| hOAT3 | Forward | GGCAGGTATACTGATTGGAG | ||
| Reverse | TCCACCAGGATGATAGGAAG | |||
| HNF1α | Forward | TGGGTCCTACGTTCACCAAC | ||
| Reverse | TCTGCACAGGTGGCATGAGC | |||
| HNF1β | Forward | TGCACAAAGCCTCAACACCT | ||
| Reverse | GTTGGTGAGTGTACTGATGC | |||
| GAPDH | Forward | AATGACCCCTTCATTGAC | ||
|
| Reverse | TCCACGACGTACTCAGCGC | ||