Cross-contamination with tamoxifen induces transgene expression in non-exposed inducible transgenic mice

Inducible transgenic mouse models that impose a constraint on both temporal and spatial expression of a given transgene are invaluable. These animals facilitate experiments that can address the role of a specific cell or group of cells within an animal or in a particular window of time. A common approach to achieve inducibility involves the site-specific recombinase ‘Cre’, which is linked to a modified version of one of various steroid hormone-binding domains. Thus, the expression of Cre is regulated such that a functional nuclear transgene product can only be generated with the addition of an exogenous ligand. However, critical requirements of this system are that the nuclear localization of the transgene product be tightly regulated, that the dosage of the inducing agent remains consistent among experimental animals and that the transgene cassette cannot express in the absence of the inducing agent. We used the Cre ER(T2) cassette, which is regulated by the addition of the estrogen antagonist tamoxifen to determine whether cross-contamination of tamoxifen between animals housed together can be a significant source of spurious results. We found that cross-contamination of exogenous tamoxifen does occur. It occurred in all animals tested. We suggest that the mechanism of contamination is through exposure to tamoxifen in the general environment and/or to coprophagous behavior. These results have important implications for the interpretation and design of experiments that use ‘inducible’ transgenic animals.

Western blot analysis of antigens specifically recognized by natural immune responses of patients with Japanese encephalitis infections.

Specific recognition of antigenic proteins of Japanese encephalitis virus (JEV) by JE patients was investigated by using non-reducing and reducing Western immunoblot analysis. Under non-reducing conditions, the profile of JEV proteins recognized comprised E (52 kDa), NS1 (45 and 41 kDa), NS3 (66.2 kDa) and NS5 (103 and 97.4 kDa). When recognition patterns of sera from JE and dengue patients were compared, only slight differences between JE and dengue sera were found (under non-reducing conditions), involving only the 66.2 kDa protein: to this protein, JE sera exhibited greater reactivity, but not in greater frequency, than did dengue sera. In contrast, cerebrospinal fluid (CSF) from JE patients showed more differences from JE sera: CSF antibody lacked recognition of the 41 kDa protein and had lower frequencies, as well as less reactivities to several other proteins. These results suggested that restricted populations of lymphocytes were localized in the central nervous system of JE patients. The effect of reducing agent (2 beta-mercaptoethanol) on the recognition patterns of those groups of sera was also analysed: the reducing agent affected all the proteins mentioned above, however, the effects were not uniform. It is proposed that JE and dengue sera may recognize different epitopes on some or all of these proteins. Such differences cannot be detected by Western immunoblot analysis, but it would be feasible to test this hypothesis using epitope mapping with synthetic peptides in a multi-pin ELISA. Analysis in this fine detail is essential for designing improved JE vaccines.