Each recombination acceptor cassette on the Platform ACE consists of a lambda phage attP site flanked by a simian virus 40 (SV40) promoter at the 5' position and an open reading frame sequence encoding the puromycin
resistance (puromycin) gene at the 3' position. This confers puromycin resistance to cells carrying the Platform ACE (Figure
1A). All ATVs (Figure 1C) encode a bacterial attB site upstream of a promoterless, secondary drug-selectable marker gene (e.g., zeocin, blasticidin, neomycin, or hygromycin),
which becomes activated by the SV40 promoter when the ATV integrates correctly via recombination between the attB site on the ATV and an attP site residing on the Platform ACE (Figure 1D). The ATV also contains the target gene cassette, which consists of the gene(s)
of interest flanked by insulators and a 5' upstream CX promoter (chicken Beta-actin promoter and CMV immediate/early enhancer).
Multiple copies of the same gene or multiple genes (e.g., heavy and light chains of an antibody) can be placed into the ATV
and loaded on to the Platform ACE. The site-specific recombination is mediated by the ACE Integrase, a proprietary version
of the lambda phage integrase that has been genetically engineered to function in mammalian cells without bacterial cofactors
(Figure 1), resulting in the generation of two new sites, attR and attL. The ACE Integrase reaction is unidirectional and catalyzes only the integration of the ATV onto the Platform ACE because
it lacks the bacterial cofactors required for excision. The combination of multiple attP sites on the Platform ACE and the unidirectional ACE Integrase enables multiple loadings (during a single transfection)
or sequential loadings (via multiple transfections) with ATVs. Moreover, the ATV itself has a considerable carrying capacity
and has been able to carry payloads exceeding 1.25 Mbp.
The ACE System targeted integration increases the efficiency of screening; only cells in which the ATV has correctly integrated
into the Platform ACE are selected. Because multiple gene copies are inserted in a single round of loading into a consistent
molecular environment, very few colonies (100–200) have to be screened to identify the high-expressing clones.