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. 2011;6(8):e23160.
doi: 10.1371/journal.pone.0023160. Epub 2011 Aug 3.

Checkpoints in adenoviral production: cross-contamination and E1A

Dagmar J Haeussler  1 , Alicia M Evangelista, Joseph R Burgoyne, Richard A Cohen, Markus M Bachschmid, David R Pimental
Affiliations

Checkpoints in adenoviral production: cross-contamination and E1A

Dagmar J Haeussler et al. PLoS One. 2011.

Abstract

Adenoviruses are widely used for overexpressing proteins in primary mammalian cells. Incorporation of the early viral gene, E1A, or viral cross-contamination can occur during amplification, and identification of these products is crucial as the transcription of unwanted genetic material can impact cell function and compromise data interpretation. Here we report methods for evaluation of contaminating adenovirus and E1 viral DNA.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Homologous recombination of HEK293 Ad5 genomic DNA with the Ad construct.
HEK293 cells contain bases 1–4345 of Human Ad serotype 5 genome, including the packaging signal (ψ) and E1 region. Overlapping Ad5 sequences in 2nd generation Ad constructs can undergo single or double homologous recombination to yield replication competent Ad.
Figure 2
Figure 2. Contamination of Ad constructs during replication.
(A) Primer locations for PCR of Ad and subsequent sequencing. Primer I and primer III ensure PCR of viral DNA by overlapping either with an affinity tag or part of the construct sequence. (B) Procedure used for sequencing Ad. (C) Sequencing results from early stage Ad amplification demonstrating contamination of the GOI.
Figure 3
Figure 3. Detection and effects of E1A in HAEC.
(A) DNA was extracted from cesium chloride purified Ad or HEK293 cells and PCR was performed with primers for E1A. Ad 3 shows E1A contamination. (B) VEGF-induced tube formation in HAEC infected with either Ad containing the GOI or Ad coding for the GOI and the contaminant E1A.
See this image and copyright information in PMC

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