Fig. 1. Structures of HAC vector constructs and transcriptional activities. (A) 7C5-basic BAC DNA contained an alphoid repeat (70 kb) sequence, a mammalian selectable maker gene (SV40-bsr), human inverted telomeres (each 1.1 kb), a loxP site, and a chloramphenicol resistance (CmR) and ampicillin resistance (AmpR) gene. Insertion vectors contained a kanamycin resistance (KanR) gene, sequences of interest (SOI) and a loxP site, from which the replication origin had been removed (see Materials and Methods). (B) HAC vector constructs were linearized with I-SceI endonuclease. Each HAC vector had an insertion of different DNA at the loxP site on the left arm of 7C5-basic BAC: 7C5-SV BAC lacks the promoter of ßgeo gene. 7C5-SV/CMV BAC contains the ßgeo gene with CMV promoter. 7C5-INS BAC possesses the chicken ß-globin insulator sequences (cHS4) flanking both sides of the CMV-ßgeo cassette. 7C5-SV/CMV_rev BAC is identical to 7C5-SV/CMV BAC but contains the CMV-ßgeo cassette in reversed orientation. (C) ß-Galactosidase activity of HT1080 cells was analyzed 24 hours after transfection of either SV-ßgeo or CMV-ßgeo plasmid DNA, or after trasfection of pBluescript II as a control. Luciferase activity of co-transfected luciferase expression vector (pRL-CMV, wako) was used for normalization. Data are the averages of three independent experiments. Error bar shows the s.e.m. (D) RT-PCR was performed 24 hours after transfection of each HAC vector DNA (7C5-SV, 7C5-SV/CMV or 7C5-INS BAC). PCR was carried out with 25 or 30 cycles against reverse transcribed cDNAs with specific primers (from top: ß-actin, bsr, bsr-pA, right junction, left junction, kanR, ßgeo-pA, ßgeo1). Control reactions were performed against mock-transcribed cDNAs without reverse transcriptase (–RT).