Checkpoints in Adenoviral Production: Cross-Contamination and E1A

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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“…Suitable cosmids were digested with PAC-1 and transfected into HEK-293 cells utilizing calcium phosphate. Adenoviral colonies were isolated and propagated in HEK-293A cells (25). Adenovirus was isolated using double cesium chloride gradient centrifugation.…”

Correction

“…We have reported elsewhere 2 the details of the methods used by which nested primer pairs were used to cover the immediate upstream adenoviral sequence and the full-length sequence of the virally expressed SERCA. The error in Tong et al was detected when the SERCA vectors were re-sequenced after discovering contamination of another pair of viral vectors being amplified in our laboratory as reported in Haeussler et al 2 As indicated in Figure 3 of Haeussler et al, we also checked for and found that the same vector intended to express only the wild type SERCA was also contaminated by the adenoviral E1A gene. Contamination by E1A can occur inadvertently by homologous recombination of the E1A gene that is present within the HEK 293 cell line and that enables amplification of the adenoviral vector which lacks this gene.…”

“…The methods are provided in full in Tong et al 1 and Haeussler et al 2 Titers of the new wild type SERCA 2b and the SERCA 2b C674S mutant were adjusted so that both increased mRNA levels 2-to 3-fold over baseline with an equal increase in SERCA protein expression confirmed by western blot. In the figure below, cultured Obese Zucker rat aorta smooth muscle cells infected with an "empty" adenoviral vector, which lacks a gene insert, migrated similarly in response to serum as the cells infected with the LacZ vector used as control in the original Figure 1b of Tong et al Cells infected with authentic wild type SERCA2b showed similar migration, but demonstrated a restoration of the ability of the nitric oxide donor, DETA NONOate, to inhibit their migration into a scratch wound (see Figure 1 below).…”

Correction

“…We have reported details of the methods used elsewhere by which nested primer pairs were used to cover the immediate upstream adenoviral sequence and the full-length sequence of the virally expressed SERCA. 2 Our finding of the error in Lancel et al 1 came when checking the SERCA vectors after discovering contamination of another pair of viral vectors being amplified in our laboratory as reported in Haeussler et al 2 We then sought an explanation of the different effects of overexpression of two SERCA C674S mutants that we realized had occurred in the experiments shown in Figure 3 of Lancel et al 1 As indicated in Figure 3 of Haeussler et al,…”

“…Methods are provided in full in Lancel et al 1 and Haeussler et al 2 In Panel A of the revised figure, BIAM-labeled SERCA2b is measured in adult rat ventricular myocytes infected with adenoviral vectors expressing LacZ, authentic wild type SERCA2b or the C674S SERCA2b mutant. As in our original report, the fraction of SERCA2b that is BIAM-labeled is decreased by HNO in LacZand WT-overexpressing cells, whereas the fraction of BIAM-labeled SERCA2b is decreased in cells overexpressing the C674S mutant, but is not decreased further by HNO.…”