Primers used to create luciferase promoter constructs and mutants
UGT1A8, -1A9, and -1A10 promoter fragments of approximately 2000, 1000, 400, 140, 80, and 25 base pairs in length were amplified by PCR using the forward primers (primers are named by gene, size of promoter fragment, and then orientation) in combination with either the 1A8/10rev, 1A10rev1, 1A9rev1, and 1A9rev2 reverse primers as described under Materials and Methods. The resulting PCR products were subsequently cloned into the pGL3-basic vector (see Materials and Methods). Mutations in the UGT1A8, -1A9, and -1A10 HNF1 site were generated using the HNF1m forward and reverse primer pairs (primers are named by gene, mutation, and then orientation) according to the QuikChange site-directed mutagenesis kit (Stratagene). Mutations in the UGT1A8 and -1A10 Cdx2 site were generated through a two-step PCR procedure. Overlapping PCR fragments were amplified using the following primer pairs: 1A8-140for and 1A8-Cdx2mrev (UGT1A8 fragment 1), 1A10-140for and 1A10-Cdx2mrev (UGT1A10 fragment 1), 1A8-Cdx2mfor and 1A8/10rev (UGT1A8 fragment 2), 1A10-Cdx2mfor and 1A8/10rev (UGT1A10 fragment 2), 1A8-140for and 1A8-Cdx2(A to C)mrev (UGT1A10* fragment 1), and 1A10-Cdx2(A to C)mfor and 1A8/10rev (UGT1A10* fragment 2). Fragment 1 of each respective gene was mixed with fragment 2 and subsequently amplified using the 1A8-140for or 1A10-140for forward and the 1A8/10rev reverse primers (*, A-to-C mutation in Cdx2 site). Mutations are bold, and the restriction sites used for cloning are underlined.
| Primer | Nucleotide Sequence |
|---|---|
| 1A8-2000for | 5′-TTTTGGTACCAGAGCTGAGTTCAGGTC-3′ |
| 1A8-1000for | 5′-TTTTGGTACCTACGTTTAAACAACCAG-3′ |
| 1A8-400for | 5′-TTTTGGTACCTTGGAACATAGGATACC-3′ |
| 1A8-140rev | 5′-TTTTGGTACCTCAAAAAATGATACTC-3′ |
| 1A8-80for | 5′-TTTTGGTACCATTTTTTTTTTTTTTATGAC-3′ |
| 1A8-25for | 5′-TTTTGGṪACCGTGCCTGTAGTTCTTCC-3′ |
| 1A9-1000for | 5′-ACGCATCTGCAGGTTCTTGCCGAAGCCTTC-3′ |
| 1A9-400for | 5′-ACGCATCTGCAGAGAGCATGAGTTGCCATC-3′ |
| 1A9-140rev | 5′-TTTTGGTACCTCAGCAAAAGCTACTC-3′ |
| 1A9-80for | 5′-TTTTGGTACCATTTTTTTTTATGAAAGGAT-3′ |
| 1A9-25for | 5′-ACGCAGGGTACCGTGCTGGTATTTCTCCCA-3′ |
| 1A10-2000for | 5′-CTATACGCGTGTATTAGGTTTGCTTGGT-3′ |
| 1A10-1000for | 5′-CTGGGGTACCTGTACTGTCGTATAC-3′ |
| 1A10-400for | 5′-CTGTGGTACCCCTGGAACATGAGATGCC-3′ |
| 1A10-140rev | 5′-AGTAGGTACCTCAGCAAATGATACTC-3′ |
| 1A10-80for | 5′-GTGAGGTACCTTTTTTTTTTTTTTTATGAA-3′ |
| 1A10-25for | 5′-TTTTGGTACCGTGCCTGTACTTCTTCC-3′ |
| 1A8/10rev | 5′-AGCCACGCGTGAACTGCAGCCCGAGCC-3′ |
| 1A10rev1 | 5′-CCACCCCGGGCGAGCCATGAGAGAACTG-3′ |
| 1A9rev1 | 5′-AGCCATCTCGAGCAGAGAACTGCAGCTGAGAGC-3′ |
| 1A9rev2 | 5′-AGCCATACGCGTCAGAGAACTGCAGCTGAGAGC-3′ |
| 1A8/10-HNF1mfor | 5′-GTTCTTATGAGTCGCTCATTGGCAGTGAGTG-3′ |
| 1A8/10-HNF1mrev | 5′-CACTCACTGCCTATGAGCGACTCATAAGAAC-3′ |
| 1A9-HNF1mfor | 5′-CTTGTTCTTTTGGGTCGCTCATTGTCAGTGACTG-3′ |
| 1A9-HNF1mrev | 5′-CAGTCACTGACAATGAGCGACCCAAAAGAACAAG-3′ |
| 1A8-Cdx2mfor | 5′-TGTGATTTTTTTTTTTTGGCTGACAGGATAAATACAC-3′ |
| 1A8-Cdx2mrev | 5′-CCTGTCAGCCAAAAAAAAAAAATCACACTCACT-3′ |
| 1A10-Cdx2mfor | 5′-TGTGATTTTTTTTTTTTTGGCTGAAAGGATAAATACAC-3′ |
| 1A10-Cdx2mrev | 5′-CCTTTCAGCCAAAAAAAAAAAAATCACACTCACT-3′ |
| 1A10-Cdx2(A to C)mfor | 5′-TGTGATTTTTTTTTTTTTTTATGACAGGATAAATACAC-3′ |
| 1A10-Cdx2(A to C)mrev | 5′-CCTGTCATAAAAAAAAAAAAAAATCACACTCACT-3′ |