Recombinational construction in Escherichia coli of infectious adenoviral genomes

Construction of incP-derived plasmids harboring recombinant Ad5 genomes. (A) Construction of pXL2689. pXL2672 is a pRK290-based incP replicon that contains the right and left Ad5 termini. The only clone obtained during pXL2672 construction contained the expected 5.1-kb EcoRI fragment with the Ad5 termini, together with an additional 4.1-kb EcoRI fragment encompassing the glnE gene of E. coli (GenBank accession no. Z21844Z21844). This plasmid was used further because the presence of this additional fragment did not alter the PacI-mediated excision of the fragment bordered by the viral ITRs. The two consecutive homologous recombinations that led to the construction of plasmid pXL2684 by gene replacement are referred to as DCO (double crossing-over) between pFG144 and pXL2672. XbaI deletion then generated plasmid pXL2689. PacI restriction of pXL2689 produced a RAd genome that contains deletions within the E1 and E3 regions. Vertical and horizontal striped arrows represent the left and right termini, respectively, and the shaded box refers to the sacB gene and the gene conferring Spr. The unshaded boxed area represents pMX2, a 2.2-kb ColE1 (pBR322) derivative carrying an ampicillin-resistance cassette. (B) Construction of pXL2822 by gene replacement (DCO) between pXL2689 and pGY63. Recombinational cloning followed by PacI restriction generates an infectious RAd genome that contains a functional CMV-nlslacZ expression cassette (black box). Vertical and horizontal striped boxes represent the left ITR and Ψ, and sequences encompassing the pIX gene, respectively. (C) Construction of pXL2789 and pXL2811. (1) Gene replacement (DCO) between the Tetr pXL2689 (incP) and Kmr pYJ6 (ColE1) plasmids generated pXL2789, an incP replicon containing an E1−E3−E4− RAd genome. The striped boxes represent E4 sequences. (2) Gene replacement between pXL2789 and pLC1 subsequently generated pXL2811, an incP-based replicon that contains a PacI-excisable E1−E3−E4− recombinant genome containing an RSV LTR/nlslacZ expression cassette (bricked box). Recombinational gene replacement between this incP replicon and pACK2 (ColE1) generates pXL3017, which is identical to pXL2811 except that a pCMV-driven HSV1-TK expression cassette replaces that of pXL2811. Solid lines represent adenoviral sequences and dashed lines refer to non-viral sequences. Crossed lines represent recombination events. The black dot represents the RK2 origin of replication (incP). (▿, deletion; X, XbaI; P, PacI.)