Figure 3

Schematic diagram of construction of the alphoid ^tetO-HAC containing the EGFP transgene to measure chromosome instability. (A) Three steps of MMCT to transfer the HAC. The original alphoid^tetO-HAC was generated in human fibrocarcoma HT1080 cells [8]. The alphoid^tetO-HAC was transferred to homologous recombination proficient chicken DT40 cells via MMCT. In chicken DT40 cells, a loxP gene loading site was generated in the HAC [13]. The modified alphoid^tetO-HAC was transferred to HPRT-deficient hamster CHO cells via MMCT. (B) Loading of the EGFP-containing cassette into the HAC was carried out in hamster CHO cells. Insertion of the cassette into the loxP site of the HAC by Cre/lox-mediated recombination is accompanied by reconstitution of the HPRT gene allowing the cells selection on the HAT medium. This modified HAC was transferred back to human HT1080 cells via third round of MMCT. (C) Fluorescence images of cells carrying the HAC with the EGFP cassette are shown. (D) FISH analysis of the HAC-containing HT1080 clone. The HAC was visualized using BAC32-2-mer(tetO) DNA containing 40 kb of alphoid-tetO array cloned into a BAC vector as described previously [8] (red). Chromosomal DNA was counterstained with DAPI. The HAC is indicated by arrowhead.