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. 1999 Sep;73(9):7835-41.

Improved Production of Gutted Adenovirus in Cells Expressing Adenovirus Preterminal Protein and DNA Polymerase

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Free PMC article

Improved Production of Gutted Adenovirus in Cells Expressing Adenovirus Preterminal Protein and DNA Polymerase

D Hartigan-O'Connor et al. J Virol. .
Free PMC article

Abstract

Production of gutted, or helper-dependent, adenovirus vectors by current methods is inefficient. Typically, a plasmid form of the gutted genome is transfected with helper viral DNA into 293 cells; the resulting lysate is serially passaged to increase the titer of gutted virions. Inefficient production of gutted virus particles after cotransfection is likely due to suboptimal association of replication factors with the abnormal origins found in these plasmid substrates. To test this hypothesis, we explored whether gutted virus production would be facilitated by transfection into cells expressing various viral replication factors. We observed that C7 cells, coexpressing adenoviral DNA polymerase and preterminal protein, converted plasmid DNA into replicating virus approximately 50 times more efficiently than did 293 cells. This property of C7 cells can be used to greatly increase the efficiency of gutted virus production after cotransfection of gutted and helper viral DNA. These cells should also be useful for generation of recombinant adenovirus from any plasmid-based precursor.

Figures

FIG. 1
FIG. 1
Infectivity of circular Ad genomes in low-passage (LP) 293 and C7 cells. (A) Qualitative comparison. Plates of low-passage 293 (left) or C7 (right) cells were transfected with 8.8 μg of pFG140 or pAd5βdys. The top set of two plates shows neutral red staining of pFG140-transfected plates 6 days after overlay. The middle set of two plates shows neutral red staining of pFG140-transfected plates 9 days after overlay. The bottom set of two fields shows X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining of pAd5βdys-transfected plates 3 days after transfection. The numbers of blue cells per unit of area are not significantly different in either cell line. (B) Quantitative comparison. Plates of low-passage 293 (n = 8) or C7 (n = 8) cells were transfected with 2.5 μg of pFG140 and stained 6 days after overlay. The graph shows the average total number of visible plaques per plate at 6 days, error bars represent the standard error of the mean, and the asterisk represents statistical significance according to the paired-samples t test.
FIG. 2
FIG. 2
Fold stimulation of plaquing efficiency in C7 cells by physical state of the Ad genome. Plates of low-passage 293 and C7 cells were transfected with the indicated form of viral DNA or infected with intact virus, overlaid, and stained with neutral red after formation of plaques. Fold stimulation in C7 cells was then calculated as the number of plaques observed on a plate of C7 cells divided by the number of plaques observed on an equivalently treated plate of 293 cells. From left to right, the bars represent transfection with pFG140; transfection with linear, deproteinized hpAP DNA extracted from virions; transfection with terminal protein (TP)-DNA complex; and infection with hpAP virus. Bars represent average fold stimulation observed over all experiments (n ≥ 3), error bars represent the standard error of the mean, and asterisks indicate that mean fold stimulation is significantly different from 1. In typical experiments, we observed an average of 8 versus 227 plaques with linear viral DNA, 13 versus 45 plaques with TP-DNA, and 8 versus 7 plaques with intact virus.
FIG. 3
FIG. 3
Production of gutted virus particles and DNA after cotransfection into low-passage (LP) 293 or C7 cells. Cells were cotransfected with 8 μg of pGEβdys gutted viral plasmid and 1 μg, 200 ng, or 40 ng of hpAP helper viral DNA. Resulting virus was harvested after lysis or, in the case of one unlysed sample, after 2 weeks. (A) Titer of gutted virus produced after lysis of cotransfected plates. Black bars show the titer of gutted virus, in blue-forming units per milliliter (bfu/ml). These results may be converted to blue-forming units per infected cell by dividing by about 2 × 106, the approximate number of cells in a 6-cm plate. Grey bars show the ratio of gutted titer to helper titer, where the latter is determined by titering lysates for alkaline phosphatase transducing units. (B) Southern analysis of viral DNA produced after cotransfection. DNA was extracted from a small amount of lysate, digested with HindIII, and analyzed by Southern blotting with a probe that overlaps the Ad packaging signal. The left end of the helper virus is the dark 6.6-kb band near the top of the panel shown; the left end of the gutted virus is the 3.4-kb band near the bottom of the panel.
FIG. 4
FIG. 4
Cotransfection of pTP- or Ad polymerase-expressing plasmids showing the effect on infectivity of pFG140 in various cell lines. Plates of low-passage (LP) 293 (A) or B6 (B) cells were cotransfected with 3 μg of pFG140 and various amounts of pBSKSII+ (pBS), pRSV-Pol, or pRSV-pTP. Plates were then overlaid with agarose and, after formation of plaques, stained with neutral red. Fold stimulation was then calculated as the number of plaques observed after cotransfection of pFG140 with a test plasmid divided by the number of plaques observed after cotransfection of pFG140 with pBS into the same cell line. The lefthand scale indicates average fold stimulation. The righthand scale indicates a calculated estimate of the total increase in infectivity obtained by the combined effect of the cotransfected test plasmid and use of the indicated cell line. The first bar in each set represents the control value of 1. The second bar in each set represents the average effect of adding 3 μg of pRSV-Pol, instead of 3 μg of pBS, to the transfection mixture. The third bar represents the average effect of 3 μg of pRSV-pTP. The fourth bar in each set shows the effect of cotransfecting 3 μg of pRSV-Pol and 3 μg of pRSV-pTP with 3 μg of pFG140. In panel A, the fifth and sixth bars show the effect of cotransfecting 1.5 or 0.75 μg of pRSV-Pol, respectively, together with 3 μg each of pFG140 and pRSV-pTP. Note that in all cases a total of 9 μg of DNA was transfected into a 6-cm plate. Error bars represent the standard error of the mean, and asterisks indicate that mean fold stimulation is significantly different from 1.

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