Recombinational construction in Escherichia coli of infectious adenoviral genomes

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 incP͞Ad5 replicon and the ColE1 plasmid. The overall procedure requires only the in vitro engineering of the ColE1-derivative by f lanking the desired modification with small stretches of identical sequences. In the first step, a cointegrate between the tetracycline-resistant incP͞Ad5 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 incP͞Ad5 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.

Transgene amplification and persistence after delivery of retroviral vector and packaging functions with E1/E4-deleted adenoviruses

“…A NotI/BamHI PCR fragment corresponding to the polyadenylation signal of the hBGH gene was amplified from pcDNA3 (Invitrogen, Groningen, The Netherlands) with an upstream primer, 5Ј-AAAAAAAAGCGGCCGCCCTAAATGCTAGAGCTC-GCTG-3Ј, and a downstream primer, 5Ј-CGCGGATC-CCCACCGCATCCCCAGC-3Ј, and cloned between the NotI and BamHI sites of pCLTKR to generate pCLTKRpA. The BglII/BamHI restriction fragment from pCLTKRpA containing the CMV/HSV-1 tk expression cassette was inserted at the BamHI site of pXL3048 16 to generate pAdCLTK. The AvrII/ EarI fragment from pEF1␣-Env, containing the env gene cassette expression, was subsequently inserted into the SalI site of pAdCLTK to generate the shuttle plasmid pAdCLTKEE.…”

“…16 Restriction analysis and partial automated sequencing (EuroSequence Gene Service, St. Malo, France) were used to check both adenoviral backbones (Fig 1).…”

“…16 Viral prestocks were aliquoted and stored at Ϫ20°C. Restriction analysis and partial automated sequencing were used to check the integrity of both adenoviruses.…”

“…16 In the first backbone, the MoMLV gag-pol cistron expressed from the human EF1␣ promoter has been inserted in place of the E1 genes (pAdGAG/POL, see Materials and Methods). In the second backbone, the 4070A amphotropic env protein expressed from the human EF1␣ promoter and the CMV/HSV-1 tk expression cassette containing the MoMLV PSI ϩ packaging sequence had been inserted in place of the E1 genes (pAdTK/ENV, see Materials and Methods).…”

New tools for the generation of E1- and/or E3-substituted adenoviral vectors

“…Screening for the recombinant virus has been facilitated by using counter-selection methods, [6][7][8] by extensively fragmenting the viral DNA complexed with the adenoviral terminal protein 9 or by using Cre-lox-mediated recombination. 3 Alternative approaches have been developed where the sequence of the recombinant adenovirus is reconstituted either in yeast 10 or in E. coli [11][12][13][14][15][16] before being transfected into 293 cells. 17 Such methods have the advantage that copies of the recombinant viral DNA are purified from clones and should therefore generate homogenous virus preparations.…”