Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein

Science

Volumn 275, Issue 5298, 1997, Pages 402-405

  Lasmézas, Corinne I ^a   Deslys, Jean Philippe ^a   Robain, Olivier ^b   Jaegly, Alexandre ^a   Beringue, Vincent ^a   Peyrin, Jean Michel ^a   Fournier, Jean Guy ^c   Hauw, Jean Jacques ^c   Rossier, Jean ^d   Dormont, Dominique ^a  

^a INSTITUT DE RADIOPROTECTION ET DE SÛRETÉ NUCLÉAIRE   (France)

^b HÔPITAL SAINT VINCENT DE PAUL   (France)

^c PITIÉ SALPÊTRIÈRE HOSPITAL   (France)

^d CNRS   (France)

Author keywords

[No Author keywords available]

Indexed keywords

PRION PROTEIN;

ANIMAL MODEL; ANIMAL TISSUE; ARTICLE; BOVINE SPONGIFORM ENCEPHALOPATHY; CATTLE DISEASE; CONTROLLED STUDY; DISEASE TRANSMISSION; MOUSE; NERVE CELL NECROSIS; NEUROLOGIC DISEASE; NONHUMAN; PRION DISEASE; PRIORITY JOURNAL; STRAIN DIFFERENCE;

ANIMALS; APOPTOSIS; ASTROCYTES; BRAIN; BRAIN CHEMISTRY; CATTLE; ENCEPHALOPATHY, BOVINE SPONGIFORM; ENDOPEPTIDASES; MICE; MICE, INBRED C57BL; NERVE TISSUE PROTEINS; PHENOTYPE; PRIONS; PURKINJE CELLS; SERIAL PASSAGE; TIME FACTORS; VACUOLES;

EID: 0344030333     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5298.402     Document Type: Article

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40. Mice were killed at the premortem stage by cervical fracture, and brains were immediately removed. One hemisphere (including the cerebellum) was frozen in liquid nitrogen and stored at -80°C for PrP analysis. (The other hemisphere was fixed for pathological examination.) For PrPres purification, the whole brain hemisphere was homogenized to 10% (w/v) in a 5% glucose solution. Briefly, proteinase K (PK) was used at 10 μg/ml (1 hour at 37°C) and digestion was blocked with phenylmethylsulfonyl fluoride (5 mM). After addition of sarkosyl to 10% and tris (pH 7.4) to 10 mM, samples were incubated for 15 min at room temperature. They were then centrifuged at 245,000g for 4 hours at 20°C on a 10% sucrose cushion (Beckmann TL100 ultracentrifuge). Pellets were resuspended in Laemmli buffer (24) and run on a 12% polyacrylamide gel. Protein immunoblotting procedures using chemiluminescence were as described (17). The standard conditions correspond to the load of samples equivalent to 4 mg of brain and a 1-min exposure time. Sensitivity of the detection can be increased by a higher loading of the gel (up to 25 mg) and a longer exposure time (up to 30 min).
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42. Thirty adult male C57BL/6 mice were injected intra-cerebrally with 20 μl of 25% BSE-infected brain homogenate. Ten control mice were injected similarly with control cow brain. Subsequent mouse-to-mouse passages used 20 μl of 10% mouse brain homogenates (corresponding to about 1/200 of a mouse brain), except for the 2PB1-1 mouse inoculum (1% homogenate). Twenty mice were injected with a 1% brain homogenate of a mouse infected with experimental mouse scrapie, strain C506M3, constituting the positive control group. Negative control mice were kept in the same room and did not develop any neurological disease. The incubation periods correspond to survival times assessed according to the criteria in (25).
43. - mice. The absence of localized PrPres deposits was confirmed by PrP immunohistochemistry.
44. Whole brain hemispheres were fixed overnight with a solution of 1% glutaraldehyde and 1% paraformaldehyde in 0.12 M phosphate buffer (pH 7.4). After 1 hour postfixation with 2% osmic acid, they were stained en bloc with uranyl acetate and embedded in Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate before examination with a Philips CM10 electron microscope.
45. We thank R. Bradley for BSE-infected cattle brain homogenate, C. Weissmann and R. H. Kimberlin for helpful discussions, and R. Rioux and J. C. Mascaro for expert animal care. We also thank P. Fritch and M. Wasowicz, as well as L. Court, who encouraged our research on TSE. Supported by a grant from D.R.E.T. (Paris).