A model to study the eects of a viral inactivator (b- propiolactone) on DNA ligation and gene expression in E. coli and Cos cells Dahmani M. Fathallah *, Khaled Zerria, M. Ridha Barbouche, Koussay Dellagi Laboratory of Immunology (The Molecular Genetics Group) Aupelf Uref LAF 301, Institut Pasteur de Tunis, PO Box 74, 1002 Le Belvedere, Tunis, Tunisia Received 17 September 1996; received in revised form 23 January 1998; accepted 10 March 1998 Abstract An experimental model to study the eects of viral inactivators on the biological properties of DNA was developed. b- propiolactone (bPL) was used in this model and its eects on ligation, transfer and gene expression of naked DNA were assessed. Evidence that bPL impairs these two major DNA functions are presented. The amounts of bPL that alter or abolish gene expression and prevent DNA cohesive ends ligation were determined. Based on these observations, it was concluded that this experimental approach could be used to study the eects on the biological properties of DNA of other inactivators used in vaccine preparations. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: b-propiolactone; DNA; Gene expression; Ligation 1. Introduction The use of mammalian continuous cell lines to pro- duce biologicals for human use has raised the issue that the residual cellular genomic DNA contained in the ®nal product might be of potential oncogenic risk [1, 2]. This issue has precluded the use of trans- formed cell lines to produce vaccines for humans. The eects of viral inactivators on DNA properties of ligation and gene expression through which a foreign DNA might randomly integrate into the host cell DNA and eventually leads to oncogenicity, have not been investigated. We have developed an exper- imental model where two expression systems, a prokar- yotic and a eukaryotic one, were used to study the eect of b-propiolactone (bPL) treatment on gene ex- pression and DNA ligation. Our model uses a closed circular and/or linear form of an expression vector with a known reporter gene. This DNA is treated with a viral inactivator, religated (the linear form) and assayed for the expression of the reporter gene in the respective expression system. The alkylating agent bPL was chosen as viral inactivator because it is commonly used in vaccine preparations [3]. It is also the most stu- died among the chemicals used as viral inactivators. Several reports have shown that bPL reacts irreversibly with a variety of macromolecules such as proteins and nucleic acids [4, 5]. It has previously been shown that bPL treatment impairs irreversibly the DNA properties of hybridization and duplication [6]. 2. Materials and methods 2.0.1. Bacterial strain E. coli JM109 rec A1, End A1, gyr A96, thi hsd R17, Sup E44, D(LacproAB), F tra D36, proAB, laciq, ZDM15 was used to host the pGEM7 plasmid (Promega-France) expressing the b lactamase gene (ampicillin resistance gene). 2.0.2. bPL treatment Plasmids DNA (pGEM7 and CDM8/CD8a) resus- pended in 10 mMTris/1 mM EDTA, pH 7.5 (TE), was treated by various concentrations (0.01%, 0.025%, Vaccine 17 (1999) 95±98 0264-410X/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S0264-410X(98)00087-5 PERGAMON * Corresponding author. Tel: 00216 1783 022; Fax: 00216 791 833.