Pgl4.10 basic vector

The goose AMH promoter reporter vectors were constructed by PCR cloning. Seven specific primers containing Kpn I and Xho I restriction enzyme cleavage sites were designed to amplify the desired promoter fragments of the geese AMH gene and the PCR products were cloned into a Kpn I- and Xho I-digested pGL4.10-Basic vector (Promega, Wisconsin, USA). The primers including restriction enzyme sites were listed in Table S5. According to the length of the vector, the plasmids were named pGL4.10-AMH1 to pGL4.10-AMH7. All the constructs were confirmed by dual-enzyme digestion and sequencing.
Mutation vectors of three binding sites for GATA4 (−778 bp, −1399 bp and −1477 bp) of the geese AMH promoter were generated using the Fast Site-Directed Mutagenesis Kit (Tiangen, Beijing, China). Mutagenic primers were listed in Table S6. The G base at the binding site was mutated into C base where the promoter region of AMH could not be bound. These mutation vectors were named pGL4.10-AMH2-GATA4-778, 1399, 1477 (referred to as M778, M1399, M1477), respectively. All the plasmids were confirmed by DNA sequencing.

We synthesized the DNA fragments using genomic DNA from patients carrying either the wild-type (allele 1) or mutant (allele 2) sequence for each SNP studied. Regions were amplified using OneTaq Polymerase (NEBiolabs, Ipswich, MA, USA) and a pair of primers (Integrated DNA Technologies, Coralville, IA, USA) designed for each sequence (Supplementary Data Table S2). The resulting PCR products were digested with specific restriction enzymes (NEBiolabs) and were cloned into the pGL4.10-basic vector (Promega, Madison, WI, USA). The constructs were all confirmed by DNA sequencing.

Primers (Table 1) containing an XhoI or BglII (Thermo) restriction site were used to amplify a series of 5′-deletion CCR7 gene promoter fragments that were connected to the plasmid PGL4.10-Basic vector (Promega, USA) carrying a firefly luciferase reporter gene. These recombinants were named PGL4-1055 (−1055 to +74 nt, relative to the transcription start site “ATG”), PGL4-725 (−725 to +74 nt), PGL4-462 (−462 to +74 nt), PGL4-243 (−243 to +74 nt) and PGL4-108 (−108 to +74 nt). Forty-eight hours after transfection with promoter vector, cells were lysed and the intracellular luciferase activity of the lysates was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The relative luciferase units were obtained by comparison to the luciferase activity of the pRL-TK plasmid (plasmid carrying a renilla luciferase report gene as an internal reference). Luminescence measurement was carried out on a luminometer (Orion II Microplate Luminometer, Berthold Detection Systems, Germany).

Mouse Il9 promoter (from −895 to +5) was subcloned into pGL4.10 basic vector (Promega). 293T cells were transiently transfected 24 h with reporter plasmids along with expression vectors^{15 (link)} for RelA, RelB, c-Rel, p50, and p52 or control vector by Lipofectamine 2000 (Invitrogen). Luciferase was measured with the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega).

The luciferase assay experiments were performed as follows^{39 (link),52} . An approximately 500 bp sized fragment of the human her2 promoter (from −414 to +78) including the S1 sequence was generated via PCR from Hela cells and inserted into a pGL4.10-basic vector (Promega, WI, USA) for construction of the wild-type (WT) plasmid. The mutant plasmid (MU) containing S3 instead of S1 was also constructed as described, and both plasmids were verified via automated sequencing.
The cultured MCF-7 cells were plated at a density of 1 × 10⁵ cells/well into a 24-well plate and incubated at 37 °C overnight. To verify the influence of the mutation on promoter activity, 400 ng WT or MU plasmids were transfected into MCF-7 cells using Lipofectamine 2000 (Invitrogen, CA, USA) as a transfection reagent. p-RL-TK vector (40 ng) was co-transfected as an internal control. To verify the effect of the small molecule ligand on her2 promoter activity, 0–800 nM cβ was mixed with the WT plasmid and transfected into MCF-7 cells. Luciferase activities were measured using a dual luciferase reporter assay system (Promega, WI, USA) according to the manufacturer′s protocol. Relative luciferase activities were acquired by normalizing the ratio of firefly luciferase activity to renilla luciferase activity of the mutant construct with that of the wild-type construct.