Dmt competent cells

A series of plasmids containing fragments with various sizes of the 5′-flanking region of the porcine Abcb1 gene were constructed in this study. All the primers used for the construction were tailed with Nhe I site (forward primers) or Hind III site (reverse primers) (Table 1). The amplified DNA fragments were digested with Nhe I and Hind III, and immediately inserted into the pGL3-basic vector (Promega, Madison, WI, United States) and sequenced. After sequencing verification, the plasmids (-1177-Luc, -777-Luc, -345-Luc, -195-Luc, +25-Luc) were extracted with an Endo-free Plasmid Mini Kit (Omega Bio-tek, Norcross, GA, United States) and quantified.
The Sp1 mutated vectors were constructed by the PCR-based site-directed mutagenesis using the Mut Express^® II Fast Mutagenesis kit (Vazyme, Nanjing, China) and Dpn? (Thermo, Rockford, IL, United States), following the manufacturer’s protocol. The -195-Luc vector containing the Sp1 binding sites was chosen as the template. The specific mutagenic primers are shown in Table 1. PCR products were amplified with the plasmids extracted from DMT competent cells (TransGen Biotech, Beijing, China) and confirmed by sequencing.

Mutagenesis was done according to the fast mutagenesis system (Transgen, Beijing, China). In brief, the mutated plasmids were amplified with two primers containing the mutations (Supplementary Table S2) using the TransStart FastPfu DNA polymerase (Transgen, Beijing, China). The PCR conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 25 cycles of 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 3 min, final extension at 72 °C for 10 min. The amplicons were subsequently digested with DMT (Transgen, Beijing, China) enzyme for eliminating the methylated parental plasmid and then purified from agrose gels. The purified products were transformed into DMT competent cells (Transgen, Beijing, China). The mutated clones selected on plates containing antibiotics were verified by sequencing.