Fig 5. Luciferase reporter assays to evaluate of the enhancer-blocking activity of the human putative CCNB1IP1 insulator.

The 1,878 bp fragment that contains the human sequences homologous to the mouse Ccnb1ip1 insulator and enhancer was cloned in the pGL4.23 [luc2/minP] vector (a) in the sense (b) and the antisense (c) orientations. Two partial fragments, a 975 bp fragment containing both putative insulator and enhancer, and a 1,207 bp fragment containing only the putative insulator, were also cloned in the sense (d, f) and the antisense (e, g) orientations. Cos7 cells were transfected with these vectors and measured for the luciferase activities. Gray and black lines represent the vector backbone and cloned fragments, respectively. Red and blue boxes represent the putative CCNB1IP1 insulator (260 bp) and enhancer (66bp), respectively. Luciferase activities relative to that of the control vector (a) are shown (mean ± sd, n = 3).