Biol. Chem., Vol. 388, pp. 349–354, March 2007 • Copyright  by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2007.040 2007/323 Article in press - uncorrected proof Transgenic mouse brains for the evaluation and quality control of BSE tests Wolfgang J. Philipp 1, *, Darlene Groth 2,3 , Kurt Giles 3 , Pavel Vodrazka 1,a , Heinz Schimmel 1 , Muriel Feyssaguet 4 , Reet Toomik 5 , Pascal Schacher 6 , Awad A. Osman 7 , Ingolf Lachmann 7 , Angus Wear 8 , Jean-Noel Arsac 9 and Stanley B. Prusiner 2,3 1 European Commission, Directorate General Joint Research Centre, Institute for Reference Materials and Measurements, B-2440 Geel, Belgium 2 InPro Biotechnology, 870 Dubuque Avenue, South San Francisco, CA 94080, USA 3 Institute for Neurodegenerative Diseases, Department of Neurology, University of California, San Francisco, CA 94143, USA 4 Bio-Rad Laboratories, 3, boulevard Raymond Poincare ´ , F-92430 Marnes la Coquette, France 5 IDEXX Laboratories Inc., Westbrook, MA 04092, USA 6 Prionics AG, Wagistrasse 29A, CH-8952 Schlieren, Switzerland 7 AJ Roboscreen GmbH, Delitzscher Strasse, D-04129 Leipzig, Germany 8 Veterinary Laboratories Agency, Whitley Road, Longbenton, Newcastle-upon-Tyne NE12 9SE, UK 9 AFSSA, 31, Av. Tony Garnier, F-69364 Lyon Cedex 07, France * Corresponding author e-mail: wolfgang.philipp@ec.europa.eu Abstract Rapid BSE tests are widely used diagnostics in veterinary medicine and more than 11 million tests are applied worldwide. The evaluation of new rapid BSE tests and the quality assurance of approved BSE tests pose a chal- lenge owing to the natural scarcity of BSE-infected bovine brainstems and regional variations in prion titer. Transgenic mice expressing bovine prion protein (Tg4092) offer an alternative approach to these problems. To determine whether BSE-infected Tg4092 mouse brains could serve as a general standard for rapid BSE tests, we inoculated Tg4092 mice intracerebrally with BSE prions, harvested brains at defined time points post- infection and analyzed cerebral hemispheres with several approved rapid BSE tests. The results show that de novo formation of the disease-causing prion protein isoform, PrP Sc , can be monitored during the course of infection. We demonstrate that BSE-infected Tg4092 mouse brains provide a renewable and controllable source of reference samples and suggest that such samples can generally Present address: State Veterinary Institute Jihlava, Rantirovska a 93, CZ-586 05 Jihlava, Czech Republic. be used for the evaluation and quality control of rapid BSE tests. Keywords: bovine spongiform encephalopathy; prion protein; rapid BSE test; transgenic mouse. Introduction Rapid tests for the post mortem diagnosis of bovine spongiform encephalopathy (BSE) or ‘mad cow disease’ are used more than 11 million times annually worldwide. In the European Union, these rapid BSE tests were eval- uated for use by the European Commission’s (EC) Insti- tute for Reference Materials and Measurements, resulting in 12 officially approved tests (EC, 2001; Philipp and Vodrazka, 2004). However, the continuing evaluation of new rapid tests for BSE prions and the quality control of approved BSE tests are confronted with two problems: the poor availability of BSE-infected bovine brainstems and the heterogeneity in prion titer along bovine brain- stems (Safar et al., 2002). Transgenic mice expressing bovine prion protein, des- ignated Tg(BoPrP q/ q )4092/Prnp 0/0 mice (Scott et al., 1999), or Tg4092 mice for simplicity, are an alternative for overcoming these problems. We therefore inoculated Tg4092 mice intracerebrally with BSE prions, harvested their brains during the infection, and subjected large numbers of cerebral hemispheres to analysis with five different rapid BSE tests. We found that such samples are generally suitable for assessing rapid BSE tests, and that de novo formation of the disease-causing prion protein isoform, designated PrP Sc , can be monitored during the course of infection. All five rapid BSE tests initially detected nascent PrP Sc in samples from mice as early as 21 to 49 days post-infec- tion (d.p.i.). The rapid rise in brain PrP Sc between 49 and 98 d.p.i. was detected by all five tests; moreover, all five tests reached saturation in the detection of PrP Sc between 147 and 220 d.p.i. Our results demonstrate that BSE-infected Tg4092 mouse brains can be used for the assessment of rapid BSE test performance and the quality control of rapid BSE tests, as well as for the standardization of secondary reference materials. Results and discussion Tg4092 mice were intracerebrally inoculated with a first- passage pool of homogenized brains of BSE-infected Tg4092 mice. Uninoculated control and BSE-infected Tg mice were housed under the same conditions and brains were harvested at specified time points. The brains were preserved in an argon atmosphere and did not show any