Oligonucleotides used for the production of promoter constructs, site-directed mutagenesis, EMSA, and quantitative PCR With regard to the oligonucleotides used for EMSA and site-directed mutagenesis, the HNF1-motif in the hOAT1 promoter is underlined. Boldface type indicates the difference in the sequence of the per and mut compared with the wild-type sequence found in the hOAT1 promoter.
Oligonucleotide | Orientation | Sequence (5′ to 3′) |
|---|---|---|
| Primers and oligonucleotides used for the cloning of 5′-flanking regions | ||
| hOAT1 | ||
| –919 | Forward | GGTACCTAATCACTTGAACCTGGGAGGC |
| –623 | Forward | GGTACCCACCCGACATTGTGTATGACG |
| –318 | Forward | GGTACCCCTGGCAACCCTTCCCAAAAGC |
| –111 | Forward | GGTACCCCTGCCCTTTATAACCACTTGG |
| +11 | Reverse | AAGCTTGGGCAGTTTTTAATCCTTGGCC |
| mOat1 | ||
| –110 | Forward | GGTACCTTGCCCTTCATTCCCAATTGG |
| +10 | Reverse | AAGCTTGGCAGCGTTTAATCTTTGTCC |
| Oligonucleotides used for the site-directed mutagenesis | ||
| Mut | Sense | GGAATCCTTGGAGGGGCGATCCGTCTGATACCAAGTCAC |
| Oligonucleotides used for the construction of EMSA probe and competitor | ||
| wt | Sense | TCCTTGGAGGGTTAATCCTTCTGATACCAAGTC |
| Per | Sense | TCCTTGGAGGGTTAATCATTAACATACCAAGTC |
| Mut | Sense | TCCTTGGAGGGGCGATCCGTCTGATACCAAGTC |
| Primers used for the quantitative PCR | ||
| mOat1 | Forward | GGCACCTTGATTGGCTATGT |
| Reverse | AGCTTAGCCCCCTCTTCTTG | |
| Mouse GAPDH | Forward | AACGACCCCTTCATTGAC |
|
| Reverse | TCCACGACATACTCAGCAC |