Oligonucleotides used for cloning, EMSA, and ChIP experiments
Primers for cloning were designed based on the CYP2C9 sequence (GenBank accession number NT_030059). Fragments for reporter constructs were PCR amplified from a CYP2C9 construct encompassing the first 1.8 kb of CYP2C9 5-flanking region [a gift from Dr. Mia Sandberg Lundblad (Lundbeck Inc., Copenhagen, Denmark), construct slightly modified]. Nonsense mutations in GATA sites I to IV were introduced into construct 2C9_−331_wt, resulting in the plasmids 2C9_−331_m1, 2C9_−331_m2, 2C9_−331_m3, 2C9_−331_m4, and 2C9_−331_m1+2, respectively. 2C9wt1+2 and 2C9wt3+4: EMSA oligonucleotides containing wild-type GATA binding site I and II or GATA binding site III and IV. 2C9mut1, 2C9m2, 2C9m3, 2C9m4, 2C9m1+2: oligonucleotides with destructive mutations in GATA binding site I, II, III, IV or I and II together. GATA binding sites are underlined. Mutations that destroy any GATA motifs are highlighted in bold. ChIP Primer set was used in ChIP analysis leading to a PCR product of 225 bp of length. The PCR product includes all four possible GATA binding sites.
| Primer Name | Sequence |
|---|---|
| Cloning primers | Wild type |
| CYP2C9_−735 fw | 5′-CAGACGCGTGCTATGAGCTGTGTGGC ACC |
| CYP2C9_−421 fw | 5′-CAGACGCGTAATATACAAGG CATAGAATAT GG |
| CYP2C9_−331 fw | 5′-CAGACGCGTCA GATTATTTACTTCAGTGCT |
| CYP2C9 rev | 5′-CAGCTCGAGTGAAGCCTTCTCTTCTTGTTAA |
| Mutant | |
| CYP2C9_mut1_fw | 5′-CAAAGGACATTTTATTTTTTTTTGTATCAGTG |
| CYP2C9_mut1_rev | 5′-AAA AATAAAATGTCCTTTGGTCTTGTTCT |
| CYP2C9_mut2_fw | 5′-GACATTTTATTTTTATCTGTTTTAGTGGGTCAA |
| CYP2C9_mut2_rev | 5′-ACAGATAAAAATAAAATGTCCTTTGGTCTT |
| CYP2C9_mut3_fw | 5′-ATATAGTGGACCTAGGTTTTTGGTCAATTT |
| CYP2C9_mut3_rev | 5′-ACCTAGGTCCACTATATGCTCCTTCTGAAA |
| CYP2C9_mut4_fw | 5′-GGTGATTGGTCAATTAAAACATCAAAGAGG |
| CYP2C9_mut4_rev | 5′-AATTGACCAATCACCTAGGTCCACTATATG |
| CYP2C9_mut1+2_fw | 5′-CAAAGGACATTTTATTTTTTTTGTTTTTAGTG |
| CYP2C9_mut1+2_rev | 5′-AAA AATAAAATGTCCTTTGGTCTTGTTCT |
| EMSA oligonucleotides (forward) | |
| 2C19wt1+2 | 5′-ACCAAAGGACATTTTATTTTTATCTGTATCAGTGGGTCAAAGTCCTTTCA |
| 2C9wt3+4 | 5′-ATATAGTGGACCTAGGTGATTGGTCAATTTATCCATCAAAGAGGCACACA |
| 2C9mut1 | 5′-ACCAAAGGACATTTTATTTTTTTTTGTATCAGTGGGTCAAAGTCCTTTC |
| 2C9mut2 | 5′-ACCAAAGGACATTTTATTTTTATCTGTTTTAGTGGGTCAAAGTCCTTTCA |
| 2C9mut3 | 5′-ATATAGTGGACCTAGGTTTTGGTCAATTTATCCATCAAAGAGGCACACAC |
| 2C9mut4 | 5′-ATATAGTGGACCTAGGTGATTGGTCA ATTAAAACATCAAAGAGGCACACAC |
| 2C9mut1+2 | 5′-ACCA AAGGACATTT TATTTTTTTT TGTTTTAGTG GGTCAAAGTC CTTTCA |
| Oligonucleotides used for ChIP | |
| Primer set | 5′-CCAA CCAAGTACAG TGAAACT |
| 5′-TTAAGACAACCATGAGCTTGCACT |
fw, forward; rev, reverse primers.