Kpni hindiii

The PCR primers were designed to hybridize at -2,012, -1,549, -1,105, -395, and -86 bp to generate the corresponding 5 -flanking deletion plasmid derivatives using the common downstream primer at +149 bp (5 deletion primers are listed in Table 1). The resulting amplicon was cloned into the pGL3-Basic vector (Pro-mega, Madison, WI) by using KpnI/HindIII (Takara Bio Inc.) enzyme sites. All the plasmids were confirmed by DNA sequencing.

Overlap extension PCR technology was used to generate site-directed mutants of the SREBP-1c promoter. Primers were designed to destroy the transcription factor binding sites by altering 3 nucleotides within each site (primer sequences are listed in Table 1). For the site mutant promoter, PCR reactions were conducted to generate 2 DNA fragments containing the designated mutations in the overlapping regions. Subsequently, 2 DNA fragments were pooled together as a PCR template to generate a full-length DNA fragment. The resulting amplicons were ligated to pGL3-basic vector after digestion with KpnI/HindIII (Takara Bio Inc.) to create mutant constructs. Each mutant construct was confirmed by DNA sequencing.