Recombinational construction in Escherichia coli of infectious adenoviral genomes - PubMed (original) (raw)

. 1997 Feb 18;94(4):1414-9.

doi: 10.1073/pnas.94.4.1414.

L Naudin, C Orsini, E Vigne, L Ferrero, A Le Roux, P Benoit, M Latta, C Torrent, D Branellec, P Denèfle, J F Mayaux, M Perricaudet, P Yeh

Affiliations

Recombinational construction in Escherichia coli of infectious adenoviral genomes

J Crouzet et al. Proc Natl Acad Sci U S A. 1997.

Abstract

A two-step gene replacement procedure was developed that generates infectious adenoviral genomes through homologous recombination in Escherichia coli. As a prerequisite, a human adenovirus serotype 5 (Ad5)-derived genome was first introduced as a PacI restriction fragment into an incP-derived replicon which, in contrast to ColE1-derivatives (e.g., pBR322 or pUC plasmids), is functional in a polA mutant of E. coli. Any modification can be introduced at will following two consecutive homologous recombinations between the incP/Ad5 replicon and the ColE1 plasmid. The overall procedure requires only the in vitro engineering of the ColE1-derivative by flanking the desired modification with small stretches of identical sequences. In the first step, a cointegrate between the tetracycline-resistant incP/Ad5 replicon and the kanamycin-resistant ColE1-derivative is selected by growing the polA host in the presence of both antibiotics. Resolution of this cointegrate is further selected in sucrose growth conditions due to the loss of a conditional suicide marker (the sacB gene of Bacillus subtilis) present in the ColE1 plasmid, leading to unmodified and modified incP/Ad5 replicons that can be differentiated upon restriction analysis. Consecutive rounds of this two-step cloning procedure allowed the introduction of multiple independent modifications within the virus genome, with no requirement for an intermediate virus. The potential of this procedure is demonstrated by the recovery of several E1E3E4-deleted adenoviruses following transfection of the corresponding E. coli-derived genomes in IGRP2 cells.

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Figures

Figure 1

Figure 1

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 _Eco_RI fragment with the Ad5 termini, together with an additional 4.1-kb _Eco_RI 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 _Pac_I-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. _Xba_I deletion then generated plasmid pXL2689. _Pac_I 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 Pac_I restriction generates an infectious RAd genome that contains a functional CMV-nls_lacZ 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 Pac_I-excisable E1−E3−E4− recombinant genome containing an RSV LTR/nls_lacZ 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, _Xba_I; P, _Pac_I.)

Figure 2

Figure 2

Southern blot analyses of virus DNA as compared with that of their corresponding **E. coli**-derived plasmid ancestors. (A) Agarose gel electrophoresis (0.8%) of _Pac_I + _Hin_dIII-restricted incP replicons and _Hin_dIII-restricted viral DNA from cell-transfected extracts. Lanes: M, 1 kb marker; 1 and 2, Ad2689 and pXL2689, respectively; 3 and 4, Ad2822 and pXL2822, respectively; 5 and 6, Ad2811 and pXL2811, respectively; and 7, pXL2789. (B) Autoradiograms from the blot obtained from the gel shown in A hybridized with probes that encompass the packaging sequence Ψ (B-1), the pIX gene (B-2), or the E4 region of Ad5 (B-3). Sizes of the expected fragments are indicated on the left.

Figure 3

Figure 3

Expression of E. coli β-galactosidase and HSV1-TK in IGRP2 and W162 cells, respectively. (A) IGRP2 cells were infected with Ad2811 and stained with X-Gal at 48 hr postinfection, as described (6). (B) Immunofluorescence analysis of Ad3017-infected W162 with a rabbit serum raised against purified HSV1-TK.

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