Fig. 2

Cloning of FAT promoter region in pGL3Basic: a FAT1 promoter (4.0Kb) was amplified by FAT1 gene specific primers flanked with restriction enzyme sites (MluI and NheI) from gDNA (human PBMCs). 4.0 kb PCR product was observed in gel electrophoresis. Lanes 1, 2 and 3 represent amplified FAT1 promoter at 4.0 kb, Lane M: Marker. b pGL3Basic (promoterless vector) digested with MluI and NheI restriction enzymes and analyzed by gel electrophoresis. Lane 1: Digested (4.8 kb linearized pGL3B), Lane 2: Undigested pGL3B. c Colony PCR was done to confirm the ligation of insert (FAT1 promoter) in pGL3Basic. FAT1 PCR product was eluted from gel electrophoresis followed by restriction digestion with MluI and NheI, ligated with digested pGL3Basic and transformation was done in E.coli strain (DH5 alpha). 8 colonies were picked and confirmed by colony PCR using vector specific primers (RV-GL). Lane1: positive control pGL3Control (SV40 promoter) (250 bp PCR product), Lane 2, 4, 5 and 6: Four colonies were found to be positive having 4.0 kb insert amplified product. d Positive colony was confirmed by double digestion with MluI-NheI. Lane1: undigested pGL3F4 vector and lane 2: Digested pGL3F4; released 4.0 kb (insert) and 4.8 kb vector backbone. e Cloned FAT1 promoter (pGL3F4 construct) digested with KpnI restriction enzyme to confirm the 5′ to 3′ directionality of insert. Lane 1: Digested pGL3F4 with KpnI released 3.3 kb insert and 5.5 kb vector backbone plus part of the insert and lane 2: Undigested pGL3F4. f A representative diagram of FAT1 construct (pGL3F4) showing position of KpnI site on vector (pGL3B) (at + 5 position) and insert [FAT1 promoter at + 105 bp w.r.t. Transcription start site (+ 1)]. g A representative diagram of the full length 4.0 kb FAT1 promoter construct (pGL3F4) that was used in cloning