Figure 1.

Principle of the adenovirus/pEPito hybrid vector and DNA sequences contained in the high-capacity adenoviral vectors to establish the hybrid-vector system. (a) Principle of the adenovirus/pEPito hybrid vector. (1) High-capacity adenoviral vectors HCAdV-pEPito-FRT² and HCAdV-FLPe simultaneously (i) transduce the target cell. (ii) FLPe is expressed from HCAdV-FLPe and recombines HCAdV-pEPito-FRT². (iii) Products of FLPe recombination are the recircularized pEPito insert containing one FRT site and the linked and linear HCAdV-backbone fragments which were flanking the pEPito insert. (2) Mitotic stability of recombined pEPito-FRT. pEPito-FRT linked to metaphase chromosomes by putative nuclear matrix proteins (X) mediates retention of the plasmid replicon. (3) Replication of pEPito-FRT. It is speculated that the prereplicative complex consisting of the origin recognition complex (orc) and the minichromosome maintenance complex (mcm) binds at multiple sites of the excised plasmid replicon pEPito-FRT. (b) DNA sequences within the adenoviral vector HCAdV-pEPito-FRT² containing the plasmid replicon pEPito flanked by FRT sites for FLPe-mediated recombination and excision.²¹ (c) The control vector HCAdV-pEPito-ΔS/MAR-FRT² contains identical DNA sequences as HCAdV-pEPito-FRT² but lacks the S/MAR sequence. Therefore, the excised plasmid is not maintained during cell division. (d) The vector HCAdV-FLPe was used in our previous study⁶ and expresses FLPe under the control of the elongation factor-1α promoter (EF1α). In addition, this vector contains an expression cassette for an inactive version of Sleeping Beauty transposase (mSB) expressed under the control of the phosphor-glycerate-kinase promoter (PGK). (e) The irrelevant vector HCAdV-hFIX was used as an infection control. As described in a previous study,²¹ this vector encodes the human coagulation factor IX (hFIX) under the control of the human α1-antitrypsin promoter (hAAT). BLA, β-lactamase gene; BSD, blasticidin resistance gene; eGFP, enhanced green fluorescence protein; FRT, recognition site of FLPe-recombinase; hCMV/EF1, elongation factor α1 promoter with human cytomegalovirus enhancer; IRES, internal ribosomal entry site; ITR, left and right inverted terminal repeats of adenovirus serotype 5; S/MAR: scaffold/matrix attachment region; stuffer DNA, stuffer DNA contained in the HCAdV DNA molecule based on the previously described HCAdV production plasmid pAdFTC.