Components of the Platform ACE System. ( A ) Platform ACE is a murine artificial chromosome pre-engineered to contain multiple recombination acceptor att P sites. ( B ) ACE Integrase is based on the enzyme lambda integrase, which has been modified as described in the text. The modification renders the integrase functionally independent of bacterial host cell factors and capable of operating in a mammalian context. In the Platform ACE system, an ACE Integrase expression vector is co-transfected with a targeting vector (see below) into a cell line harboring the Platform ACE. The transiently expressed ACE Integrase then catalyses the integration of the targeting vector onto the Platform ACE. ( C ) The ATV is a plasmid-based shuttle vector that conveys a gene(s) of interest onto the Platform ACE by means of targeted recombination between the recombination acceptor att P sites present on the Platform ACE and the recombination donor att B site of the ATV, catalyzed by the ACE Integrase. The presence of a promoterless antibiotic resistance gene downstream of the att B donor site allows for selection of targeted integration events. ( D ) A representation of a 'loaded' recombination acceptor site on the Platform ACE. Note that the targeted integration of the targeting vector has resulted in the activation of the promoterless antibiotic resistance gene by virtue of its in frame insertion downstream of the SV40 promoter present in the ACE acceptor site. ( E ) Southern-blot analysis of Platform ACE. Genomic DNA was hybridized with a labeled probe encoding the SV40 promoter and att P site. Lanes 1–3: copy number controls: 50 copies (lane 1), 100 copies (lane 2), 250 copies (lane 3); LMTK − negative control (without ACE, Lane 4) and CHR1 cell line (containing Platform ACE, Lane 5). See Materials and Methods for details.
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