143Chromosome Engineering with Lambda-Integrase Mediated Recombination System
Site-specifc loading o a transgene onto the Platorm ACE
) The satellite DNA-based artifcial chromosome gener-ated by large-scale amplifcation contains multiple integration sites. (
) The recombination acceptor sites or the ACE-integrase, attP is located between a selectable marker gene and its promoter. (
) The ACE targeting vector (ATV) carries agene o interest bordered with insulator sequences, and the attB integrase specifc site upstream a promoterless select-able marker gene. (
) Expression o ACE-integrase catalyzes the recombination between attP and attB sites resulting in thesite-specifc integration o ATV into Platorm ACE. Thus, the promoterless selectable marker gene o ATV will be driven bythe promoter on Platorm ACE and drug resistance will be provided by the integrated marker gene o ATV.Fig. 2. Detection site-specifc integration in targeted cell lines by PCR. PCR was carried out using 193AF as the orwardprimer specifc to Platorm ACE and a reverse primer specifc to the selectable marker gene o ATV; the templates weregenomic DNAs o dierent transormed clones. Site-specifc targeting to Platorm ACE was detected in the genome oclones represented in lanes 1, 3, 4, and 5. In lane 2, the PCR showed no site-specifc integration o ATV. Lane 6 is thepositive, and lane 7 is the negative control o PCR, respectively. The Marker is the 1 kB DNA ladder (Fermentas).