FIG. 2.

Identification of the Cnot2 gene by screening with the piggyBac vector as a positive regulator of conversion from partial to genuine iPSCs. (A) Protocol for the piggyBac-mediated activation screen. A piggyBac vector bearing the murine stem cell virus (MSCV) enhancer/promoter [20] and a transposase expression vector were introduced into partial iPSC clone 2B1 by lipofection. Changes of the culture conditions including hygromycin selection (600 μg/mL) were performed as indicated. (B) Bright field and fluorescence images of three GFP-positive clones yielded by the piggyBac-mediated activation screen. (C) Integration sites of the vector in the three GFP-positive clones. Splinkerette PCR to determine the integration sites of the vector was performed as described by Guo and Smith [20]. (D) Diagram of insertion of the piggyBac vector in the first intron of the Cnot2 gene. Open and solid red boxes represent noncoding and coding exons, respectively. Blue and red boxes in the piggyBac vector indicate the hygromycin-resistance gene and MSCV enhancer/promoter, respectively, while gray arrows represent the 5′ and 3′ long terminal repeats of the vector. (E) Identification of the Cnot2 transcript carrying a part of the 3′ long terminal repeat sequence of the piggyBac vector.