Construction of the iHAC for reprogramming human somatic cells. (A) Schematic diagram of the iHAC/X53. iHAC/X53 is carrying the expression cassettes for multi copies of a set of four reprogramming factors (2 copies of Klf4, c-Myc and Sox2, and also 4 copies of Oct4), a human P53shRNA construct and DsRed (R). (B) Expression of the four reprogramming factors contained in the iHAC vectors was confirmed by qRT-PCR. Transcript levels of transgenes were standardized to Gapdh. Transcript levels in CHO/iHAC/X53 were compared to levels in CHO/iHAC2/mp25. Error bars, s. d. (C) FISH analysis of CHO/iHAC/X53. Digoxigenin-labeled alphoid satellite marker (red) was used to detect the HAC backbone. Biotin-labeled pPAC-2CAG-O2hP53sh (green) was used to detect the reprogramming cassette in the iHAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate the iHAC vector and the insets show enlarged images of the iHAC.