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Link to original content: https://pubmed.ncbi.nlm.nih.gov/9482916
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. 1998 Mar 3;95(5):2509-14.
doi: 10.1073/pnas.95.5.2509.

A simplified system for generating recombinant adenoviruses

T C He  1 , S Zhou, L T da Costa, J Yu, K W Kinzler, B Vogelstein
Affiliations

A simplified system for generating recombinant adenoviruses

T C He et al. Proc Natl Acad Sci U S A. .

Abstract

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.

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Figures

Figure 1
Figure 1
Schematic outline of the AdEasy system. The gene of interest is first cloned into a shuttle vector, e.g., pAdTrack-CMV. The resultant plasmid is linearized by digesting with restriction endonuclease PmeI, and subsequently cotransformed into E. coli BJ5183 cells with an adenoviral backbone plasmid, e.g., pAdEasy-1. Recombinants are selected for kanamycin resistance, and recombination was confirmed by multiple restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell lines, e.g., 911 or 293 cells. Recombinant adenoviruses typically are generated within 7–10 days. The “left arm” and “right arm” represent the regions mediating homologous recombination between the shuttle vector and the adenoviral backbone vector (see Materials and Methods for sequence composition). An, polyadenylation site; Bm, BamHI; RI, EcoRI; LITR, left-hand ITR and packaging signal; RITR, right-hand ITR; Sp, SpeI.
Figure 2
Figure 2
Shuttle vectors and adenoviral plasmids. Abbreviations are defined in the legend to Fig. 1. See text for details.
Figure 3
Figure 3
Generation of stable recombinants in bacterial cells. (A) DNA from recombinant pAdEasy-GFP+GAL constructs derived from homologous recombination of pAdTrack-CMV-βgal and pAdEasy-1in BJ5183 cells was purified from minipreps. The DNA was analyzed in supercoiled form by electrophoresis through an 0.8% agarose gel and ethidium bromide staining. Lane 1, pAdEasy-1 control; lane 2, pAdTrack-GFP+GAL control; lanes 3–12, different pAdEasy-GFP+GAL clones. Based on the migration rates, the clones in lanes 3, 4, 6, 8, 9, 11, and 12 were potential valid recombinants. (B) Representative digestions with BamHI (lanes 1–3), PacI (lanes 4–6), and SpeI (lanes 7–9). Plasmids pAdTrack-CMV (lanes 1, 4, and 7), pAdEasy-1 (lanes 2, 5, and 8) and a pAdEasy-GFP+GAL recombinant (lanes 3, 6, and 9) are shown. ∗ indicate the diagnostic fragments obtained with each enzyme.
Figure 4
Figure 4
Adenovirus-producing foci after transfection of 293 cells. PacI-digested pAdEasy-GFP-GAL was transfected into 293 cells and GFP expression was visualized by fluorescence microscopy at the indicated times thereafter. Comet-like adenovirus-producing foci became apparent at 4–5 days. No such foci were observed in the cells transfected with circular (i.e., not PacI-digested) pAdEasy-GFP-Gal.
Figure 5
Figure 5
Adenoviral titer monitored by GFP expression. Linearized pAdEasy-GFP+GAL was transfected into 293 cells as described in Fig. 4, and cells were harvested at the indicated times after transfection. Two percent of a freeze/thaw lysate of these cells was used to infect 293 cells, and fluorescence microscopy of the infected cells was performed 24 hr later. No viruses were generated in 12 days after the transfection of circular (i.e., not cleaved with PacI) pAdEasy-GFP+GAL (labeled “control”).
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