University of California

Glenn C Telling

Stanley B Prusiner

Michael R Scott

Fred Ehrenkranz Cohen

Michael Glenn Scott

DNA constructs are provided of epitope-tagged proteins or protein fragments which are conveniently purified with immunoaffinity chromatography such as epitope-tagged prion proteins (PrP). Transgenic animals expressing an epitope-tagged protein are provided, including transgenic animals expressing epitope-tagged PrP. Methods for distinguishing between the conformational shapes of a protein and a convenient method for isolating a tagged protein by immunoaffinity chromatographic methods are provided.

Recombinant construct encoding epitope tagged PrP protein

Transgenic animals expressing artificial epitope-tagged proteins

Transgenic animals are produced which animals have (1) their endogenous PrP gene ablated; and (2) have an exogenous PrP gene from a genetically diverse animal. The transgenic animal is preferably a mouse, rat or hamster with mice being particularly preferred. The exogenous PrP gene is preferably from a sheep, cow, or pig with cow PrP genes being particularly preferred. When a mouse of the invention is inoculated with a sample containing prions which generally only infects a genetically diverse species (e.g. a cow) the mouse will become ill within about 250 days or less. Methods of producing the transgenic animals are disclosed including (1) microinjecting a mouse egg (having an ablated endogenous PrP gene) with a bovine PrP gene, or (2) breeding a mouse with an ablated PrP gene with a mouse with a bovine PrP gene. Mice produced are used to test samples for the presence of prions which generally only infect cows.

Detecting cow, sheep and human prions in a sample and transgenic mice

The invention includes an artificial PrP gene, a transgenic animal containing a PrP gene of another animal or the artificial PrP gene, a hybrid non-human mammal with an ablated endogenous prion protein gene and exogenous prion protein gene, assay methodology which uses the animals to detect pathogenic prions in a sample or diagnose a cause of death and standardized prion preparation used in the assay. The genome of a host animal (such as a mouse), is manipulated so that the animal is rendered susceptible to infection with prions which normally would infect only a genetically diverse test animal (such as human, cow or sheep). Pathogenic prions in a sample can be detected by injecting the sample to be tested into a mammal of the invention which has been genetically manipulated so as to be susceptible to infection from prions in the sample. Mammals which are not inoculated with the sample and others inoculated with a standardized prion preparation of the invention are used as controls in the assay to detect prions in samples which cause diseases. For example, Creutzfeldt Jakob Disease (CJD) is a fatal neurodegenerative disease of humans caused by prions. The mammals disclosed herein develop the adverse effects of such prions in a relatively short time after being inoculated with human prions.

Method of detecting prions in a sample and transgenic animal used for

The invention includes an artificial PrP gene, a transgenic animal containing a PrP gene of another animal or the artificial PrP gene, a hybrid non-human mammal with an ablated endogenous prion protein gene and exogenous prion protein gene, assay methodology which uses the animals to detect pathogenic prions in a sample and standardized prion preparation used in the assay. The genome of a host animal (such as a mouse), is manipulated so that the animal is rendered susceptible to infection with prions which normally would infect only a genetically diverse test animal (such as human, cow or sheep). A PrP gene of the host is preferably manipulated to include a mutation which matches a mutation which causes prion disease in the genetically diverse mammal. Pathogenic prions in a sample can be detected by injecting the sample to be tested into a mammal of the invention which has been genetically manipulated so as to be susceptible to infection from prions in the sample. Mammals which are not inoculated with the sample and others inoculated with a standardized prion preparation of the invention are used as controls in the assay to detect prions in samples which cause diseases. For example, Creutzfeldt Jakob Disease (CJD) is a fatal neurodegenerative disease of humans caused by prions.

Detecting prions in a sample and prion preparation and transgenic animal

The invention includes an artificial PrP gene, a transgenic animal containing a PrP gene of another animal or the artificial PrP gene, a hybrid non-human mammal with an ablated endogenous prion gene and exogenous prion gene and assay methodology which uses the animals to detect pathogenic prions in a sample or diagnose a cause of death. The artificial gene includes a sequence such that when it is inserted into the genome of a host animal (such as a mouse), the animal is rendered susceptible to infection with prions which normally would infect only a genetically diverse test animal (such as human, cow or sheep). The artificial PrP gene may be comprised of a completely artificial polynucleotide sequence. Alternatively, the artificial gene may be comprised of the codon sequence of a host animal with one or more codon substitutions being made wherein the substitutions are preferably corresponding PrP gene codons from a genetically diverse test animal. Pathogenic prions in a sample can be detected by injecting the sample to be tested into a transgenic mouse which includes the different codons of the prion protein gene of the animal (e.g. human) in danger of infection from prions in the sample.

Prions are protein based infectious material that cause of variety of diseases such as Scrapie, bovine spongiform encephalopathy (also known as 'Mad Cow' disease), Creutzfeldt Jakob Disease, Kuru, and fatal familial insomnia. The invention is directed to artificial prion genes that are made up of elements of the prion genes of a host and test species. When these artificial prion genes are inserted into a transgenic mouse, the resultant mouse becomes susceptible to infection with prions that infect the test species but do not normally infect mice. The transgenic animals are useful for testing for the presence of prions in a sample.

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San Francisco, CA, United States of America

Glenn C Telling