WO1994024266A1 - Modeles animaux transgeniques pour la maladie d'alzheimer - Google Patents

Modeles animaux transgeniques pour la maladie d'alzheimer Download PDF

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WO1994024266A1
WO1994024266A1 PCT/US1994/004026 US9404026W WO9424266A1 WO 1994024266 A1 WO1994024266 A1 WO 1994024266A1 US 9404026 W US9404026 W US 9404026W WO 9424266 A1 WO9424266 A1 WO 9424266A1
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sequence
transgene
coding sequence
act
mammal
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PCT/US1994/004026
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Cho-Yau Yeung
Susan R. Ross
Robert D. Beech
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Board Of Trustees Of The University Of Illinois
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0312—Animal model for Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • the brains of AD patients show characteristic morphological and histological changes including cortical atrophy, loss of neurons, the presence of neurofibrillary tangles, senile neuritic plaques and diffuse amyloid plaques.
  • Neurofibrillary tangles are abnormal neurons in which the cytoplasm is filled with bundles of submicroscopic filamentous structures that are wound around each other in a helical fashion. Katzman, New Engl. J. Med. 314:964-973 (1986). A senile
  • - l - neuritic plaque is composed of a cluster of degenerating nerve terminals, both dendritic and axonal, with a core that contains extracellular arrays of fine filaments that consist of amyloid protein. Katzman, New Engl J. Med. 314:964-973 (1986). In over two-thirds of the cases examined, amyloid has been found in blood vessels of the meninges, cerebral cortex, and the hippocampus. Within the brains of patients with AD, as well as patients with Down's Syndrome (who tend to develop full-blown AD type neuropathological symptoms at a relatively early age), diffuse amyloid plaques and the amyloid cores of predominantly neuritic senile plaques contain deposits of the ⁇ lAA peptide.
  • senile plaques also contain ubiquitin, Hardy et al, Trends in Pharm. Sci. 12:383-388 (1991), the tau protein, Lee et al, Science 251:675-678 (1991), and the protease inhibitor ⁇ l-antichymotrypsin (ACT), Abraham et al, Biotechnology 7:147-153 (1989); and Rozemuller et al, Neurosci. Lett. 119:75-78 (1990).
  • the brain atrophy commonly associated with AD is found primarily in the gyri of the association areas of the cerebral cortex with relative sparing of the primary motor, somatosensory and visual cortices.
  • Terry et al. Senile Dementia of the Alzheimer type: defining a disease in Neurology of Aging, Katzman et al eds. , F.A. Davis, Philadelphia, pp. 51-84, (1983).
  • Brains from patients with Alzheimer's show no change in the number of glial cells, the smallest of the cortical cells (less than 40 ⁇ m 2 in area), although some astrocytes appear to be fibrous.
  • the number of small neurons (40 ⁇ m 2 to 90 ⁇ m 2 ), the most numerous cells in the cortex, shows no change, nor is any change seen in the number of cells in the hippocampus, the entorhinal cortex, the locus ceruleus and the nucleus basalis. Katzman, New Engl.
  • amyloid plaques cause the neuronal damage and cell loss that lead to dementia in AD. Hardy et al , Trends in Pharm. Sci. 12:383-388 (1991). Interestingly, such plaques are also usually found, albeit in much lower numbers, in the brain of normal elderly subjects. It is thus likely that amyloid plaque formation is a normal sequelae of aging, but that the process is somehow accelerated or exacerbated in AD and in Down's Syndrome.
  • ⁇ /A4 polypepti.de As suggested above, a major component of the plaques seen in AD is the self-aggregating and highly insoluble 0-amyloid A4 polypepti.de ( ⁇ /A4) which contains 39 to 43 amino acids and has a molecular weight of approximately 4.2 kD. Glenner, Cell 52:307-308 (1988); Glenner et al, Biochem. Biophys. Res. Con ⁇ n. 120:885-890 (1984); and Masters et al, Proc. Nat'l Acad. Sci. USA 82:4245-4249 (1985).
  • APP amyloid precursor protein
  • the ⁇ lAA peptide sequence is located within APP astride the junction of the trans-membrane and extracellular domains. Normally, APP can be cleaved, presumably at the cell surface, to yield secreted forms which are truncated at the carboxy terminus. Sisodia et al. , Science 248:492-495 (1990); and Esch et al. , Science 248:1122-1124 (1990).
  • the normal cleavage site is located within the 0/A4 peptide sequence, implying that production (via proteolytic cleavage) of these secreted forms of APP could preclude the formation of the j8/A4 peptide.
  • Rabin et al, Somat. Cell and Mol Genet. 12:209-216 (1986) have mapped the ACT gene to human chromosome 14 and it is interesting to note that one Canadian family displaying a form of inherited Alzheimer's disease showed genetic linkage of the disease with a region of chromosomal 14 in which the ⁇ l- antichymotrypsin gene resides. Rabin et al, supra and Weitkarp et al, Am. J. Human Genet. 35:443-453 (1983). It is possible that an abnormal accumulation of ACT could interfere with the normal proteolytic degradation of APP which would otherwise preclude the formation of the ⁇ lAA polypeptide.
  • the APP thus accumulated in the brain of a patient with AD could then be degraded via an alternative pathway that could liberate the S/A4 polypeptide which may bind to ACT and form amyloid plaques.
  • Support for the possibility that elevated protease inhibitor expression may lead to increased formation of amyloid deposits comes from the observation that the principal component of the vascular amyloid in the Icelandic form of hereditary cerebral amyloidosis is a variant of the cysteine protease inhibitor cystatin. Ghiso et al , Proc. Nat'l Acad. Sci. USA 83:2974-2978.
  • AD pathogenesis and treatment of AD have been hampered by the lack of a naturally occurring animal model system for AD.
  • Cell culture systems may be of some utility insofar as investigations into the biochemical and molecular components of AD.
  • the ideal model system would be a whole animal system in which the pathophysiological, histopathological, biochemical, and molecular basis as well as the behavioral manifestations of AD may be studied. Additionally, a whole animal system would be particularly useful in assessing various potential treatment modalities for AD and for investigating potential prophylactic measures for the prevention of AD.
  • Transgenic animals represent one possible approach to the production of an animal model system for AD.
  • Transgenic animals carry a gene which has been introduced into the germline of the animal, or into the germline of an ancestor of the animal, at an early (usually one cell) developmental stage.
  • Three basic approaches have been used to successfully introduce foreign DNA into the germline of animals. The most common involves the direct microinjection of DNA fragments into the pronucleus of a one-cell embryo which is then placed in the oviduct of a foster mother.
  • cleavage stage embryos are exposed to a recombinant retrovirus in vitro after which they are placed into the uterus of a foster mother.
  • ES cells pluripotent embryonic stem cells
  • the genetically altered cells can then be introduced into the embryonic blastocoel after which they can colonize the embryo and ultimately contribute to the germline.
  • ES cells pluripotent embryonic stem cells
  • Transgenic animals have been useful in the study of gene regulation in vivo, in the investigation of the role of certain genes in animal development, as models for the investigation of disease and for the study of various approaches to the correction of genetic diseases.
  • Palmiter et al Cell 29, 701 (1982) describes transgenic mice containing a thymidine kinase gene fused to a heavy metal-inducible metallothionein promoter sequence. Palmiter et al , Science 222, 809 (1983) describes transgenic mice containing the human growth hormone gene fused to a heavy metal-inducible metallothionein promoter sequence.
  • transgenic animal model systems for human disease represents a particularly promising approach to the understanding of the underlying pathophysiological processes of a disease as well as a promising vehicle for testing new treatments for disease.
  • Kuehn et al. Nature (London) 326:295-298 (1987)
  • Hooper et al Nature (London) 326:292-295 (1987)
  • established animal models for Lesch-Nyhan syndrome a disease involving purine metabolism which results from a mutation in the hypoxanthine-guaninephosphoribosyl transferase gene.
  • Greaves et al Nature (London) 343:183-185 (1990) described a transgenic mouse model of sickle cell disorder.
  • U.S. Patent No. 5,175,383 to Leder et al describes a transgenic animal model for benign prostatic disease.
  • the transgene in these animals comprise an int-2 gene which is operably linked to a promoter effective for the expression of the int-2 gene in the urogenital tissues of the animal.
  • the animal may have utility as a test system for agents suspected of promoting prostatic hyperplasia and as a system for testing procedures for treating or diagnosing benign prostatic hyperplasia or hypertrophy.
  • RNA expression was specific to the brain as shown by in situ hybridization.
  • the data in Quon do not allow the determination of whether or not the diffuse plaques seen in the transgenic animals contain the entire APP protein (or only the ⁇ /A4 peptide), whether an overexpression of the protease inhibitory domain alone was sufficient to cause amyloid plaque formation, or whether the deposits result from overexpression of the
  • Transgene constructs used in these studies comprised four human APPs (APP 695, APP 751, and mutated APP 695, and 751 carrying a Val to He mutation found in some cases of familial AD) fused with a vector derived from the neuron-specific enolase gene.
  • the authors assertedly detected expression of these four constructs based on Northern and Western blot analysis and by immunostaining of brain sections using human APP antibody.
  • transgene construct comprising a human ACT cDNA fused with a vector from the murine glial fibrillary acidic protein gene (GFAP) and claim that the GFAP vector effectively directs the inducible and astrocyte-specific expression of hybrid genes in vivo.
  • GFAP-ACT containing mice Six separate lines of GFAP-ACT containing mice were said to have been established but no data describing expression of the transgene construct in animals or whether these animals showed any brain histopathology were reported in the abstract.
  • AD Alzheimer's disease
  • the present invention is directed to a transgenic non-human mammal all of whose germ cells and somatic cells contain a transgene introduced into the mammal or an ancestor of the mammal.
  • the transgenes of the present invention comprise regulatory sequences and a coding sequence.
  • the regulatory sequences of the transgene are capable of directing predominantly neuronal expression of the coding sequence in the brains of transgenic animals.
  • the coding sequences of the present invention are selected from the group consisting of sequences coding for the ⁇ lP_ peptide and the human ⁇ l-antichymotrypsin (ACT).
  • Preferred regulatory sequences of the present invention comprise a 2.7 kb upstream sequence of the murine adenosine deaminase gene (ADA) and the SV40 polyadenylation sequence.
  • transgenes for use according to the present invention include a first transgene construct comprising a 2.7 kb upstream regulatory sequence of the murine ADA gene located upstream (5') to an amyloid precursor protein (APP) leader sequence which in turn is fused to the 5' end of the ⁇ l A peptide coding region, which lies 5' to an SV40 polyadenylation sequence (S/A4 transgene construct).
  • Another transgene construct useful in the present invention comprises the 2.7 kb upstream regulatory sequence from the murine ADA gene located upstream (5') to the entire coding sequence of the human ⁇ l-antichymotrypsin gene which in turn lies 5' to the SV40 polyadenylation sequence (ACT transgene construct).
  • the present invention is also directed to an autonomously replicating plasmid comprising the 0/A4 transgene construct of the present invention.
  • the present invention is directed to an autonomously replicating plasmid comprising the ACT construct of the present invention.
  • the present invention is also directed to the use of other transgenes which code for proteins (such as those containing Kunitz protease inhibitor sequences) which may alter the normal proteolytic processing of the amyloid precursor protein.
  • Another aspect of the present invention is directed to test systems using the transgenic animals of the present invention for the testing of potential therapies for the treatment or prevention of Alzheimer's disease.
  • the present invention is also directed to the establishment of cell culture systems using cells derived from the transgenic animals of the present invention, or by transfection with the transgene constructs of the present invention.
  • Figure 1 graphically illustrates the 3/A4-containing transgene present in plasmid pADA/5AP+Ll;
  • Figure 2 graphically illustrates the ACT-containing transgene present in plasmid pADAhACT
  • Figure 3 presents a Southern blot analysis of DNA from transgenic animals containing the ACT transgene
  • Figure 4 presents a Southern blot analysis of DNA from transgenic animals containing the ⁇ lP ⁇ containing transgene
  • Figure 4 presents an agarose gel showing the results of reverse transcriptase polymerase chain reaction analysis of RNA from the brains of animals containing the ACT transgene;
  • Figure 6 presents photomicrographs of in situ hybridization analysis of the expression of the ACT transgene in transgenic mouse brain (Panels A-C) and from a wild type mouse (Panel D);
  • Figure 7 presents photomicrographs of silver stained brain sections from transgenic mice expressing the ACT transgene (Panels A-E), and from a control mouse lacking the ACT transgene (Panel F);
  • Figure 8 presents photomicrographs of immunolabeled ⁇ l- antichymotrypsin containing lesions in the brain of a transgenic mouse expressing the human ACT gene (Panels A-D) and a human patient with AD (Panel E-F); and Figure 9 (Panels A-C) presents photomicrographs of immunolabeled ⁇ lAA containing lesions in the brain of the transgenic mouse expressing the human ACT gene.
  • transgenic animal models for AD The general strategy employed in the generation of the transgenic animal models for AD was to construct transgenes for the targeted expression of the (8/A4 peptide and ACT in the brain of transgenic animals. Both transgenes were constructed using a pTZ19U (U.S. Biochemicals, Cleveland Ohio) based expression vector as described by Rauth et al, Somat. Cell Genet. 16:129-141 (1990), which comprises a 2.7 kb upstream fragment of the murine adenosine deaminase gene (ADA) which was shown to direct the expression of a downstream gene in the brains of transgenic mice.
  • pTZ19U U.S. Biochemicals, Cleveland Ohio
  • ADA murine adenosine deaminase gene
  • the transgene construct comprising the 3/A4 peptide coding sequences also included a sequence coding for the 17 amino acid APP leader sequence fused to the 5' end of the 3/A4 peptide coding sequence.
  • the transgene construct comprising the human ACT gene including its own leader sequence also contained the 2.7 kb upstream sequence from the mouse ADA gene located 5' to the ACT coding sequence. Both constructs contained an SV 40 polyadenylation sequence located 3' to the respective coding sequences.
  • Transgenic mice were generated by microi ⁇ jection of the appropriate transgene into single-cell mouse embryos according to the method described by Ross et al, Proc. Nat'l Acad. Sci. USA 82:5880-5884 (1985). Potential founder mice were then analyzed for the presence of the transgene by Southern blot analysis. Founder mice were tested for their ability to transmit the transgene to their offspring by breeding to non-transgenic mice. Transgenic offspring were identified using Southern blot analysis. Transgenic offspring of the founder mice were then analyzed for expression of the transgenes in brain by in situ hybridization (for the ACT transgene) and by reverse transcriptase-polymerase chain reaction. Finally, transgenic offspring were analyzed by histologic and immunohistologic techniques for the presence of protein deposits resulting from over-expression of the respective transgenes.
  • the transgene construct containing the ⁇ amyloid peptide (S/A4) sequence was prepared by synthesizing DNA fragments corresponding to the 17 amino acid leader and 43 amino acid 3-amyloid peptide ((8/A4) portions of the human amyloid precursor protein (APP), in such a way that the 3' end of the leader sequence overlapped with the 5' end of the gene fragment encoding the /S/A4 peptide. Kang et al , Nature 325:733-736 (1987).
  • the two fragments were then be annealed and extended using the splicing by overlap extension (SOE) method described by Horton et al, BioTechniques 8:528-535 (1990) to create a new artificial gene encoding a single polypeptide comprising the leader sequence of APP fused to the /3/A4 peptide with all of the intervening sequences, and all of the downstream sequences, deleted.
  • SOE overlap extension
  • the APP leader sequence was synthesized as overlapping oligonucleotides, which were then annealed to each other and filled in under PCR conditions described below, and then joined to a S/A4 peptide sequence obtained initially by SOE. The fused product was then cloned into the expression vector described above and in Rauth et al. , supra.
  • the primer sequences used were designed based on Kang et al. , supra and were modified to incorporate a Bam HI cloning site, and three redundant bases at 5' end of each primer, overlap to allow joining to /S/A4 region.
  • the sequences used were as follows:
  • primer sequences were:
  • PCR was carried out in a 100 ⁇ reaction volume containing 2 units of Taq DNA polymerase (Promega Biotech, Madison, Wisconsin) (DNA) in a 1.5 mM MgCl 2 , 0.2 mM dNTPs (dATP, dTTP, dCTP and dGTP), reaction buffer containing 50 ⁇ of non-purified DNA from heat denatured phage stock (from a ⁇ gtlO human testis cDNA library) and 10 pmol of each of primer /SAP A and /SAP B.
  • Amplification was carried out, after incubating for 5 min. at 94°, via 35 cycles of (1) 1 min. at 94°; (2) 1 min. at 60°; (3) 1 min. at 72°; followed by 10 min. at 72°; and storage at 4°,
  • the SOE product was then separated on a 3% agarose/TBE gel and stained with 0.5 ⁇ g/ml ethidium bromide (EtBr).
  • the band corresponding to the SOE product was excised from the gel and DNA was isolated using a 0.45 ⁇ m spin filter, (Millipore Bedford, MA) ethanol precipitated and resuspended.
  • An aliquot of the isolated DNA was then cut with the restriction enzyme Bam HI and cloned into the Bam HI site of the expression vector described above in Rauth et al , supra in which the 3' cloning site had been deleted (pDT-1).
  • the orientation of the inserted DNA was determined by restriction digests and the sequence of the inserted DNA was determined by dideoxy sequencing using a kit from U.S. Biochemicals (Cleveland, Ohio) according to the manufacturers' instructions.
  • the new primer sequences used to synthesize the leader sequence were:
  • ⁇ AP A2 5'ATAGGATCCATGCTGCCCGGTTTGGCACT 3'; (SEQ ID NO. 5) ⁇ AP B 5 'ATTCTGC ATCCGCCCGAGCCGTCC AGGCGG 3 ' ; (SEQ ID NO. 2).
  • ⁇ AP B2 5'ATTCTGCATCCGCCCGAGCCGTCCAGGCCGG 3' (SEQ ID NO. 6)
  • the three primers were annealed and extended under PCR conditions in a 50 ⁇ reaction volume containing 2 units of Taq DNA polymerase (Promega Biotech) in a 1.5 mM MgCl 2 , 0.2 mM dNTPs reaction buffer containing 10 pmol of each primer and 2 ⁇ l of heat denatured phage stock. After incubating for 2 min. at 94°, amplification was carried out in a Coy Tempcycler via 35 cycles of (1) 1 min. at 94°, (2) 1 min. at 55°; (3) 1 min. at 72°; followed by 10 min. at 72°; and storage at 4°.
  • Primer sequences used to amplify the ⁇ amyloid encoding sequences were:
  • /SAP D2 5'ATAGGATCCTCACGCTATGACAACACCGCCCA 3' (SEQ ID NO. 7).
  • Sequences encoding the ⁇ amyloid peptide were amplified from one of the clones originally obtained, (ps/SAPl) in a 50 ⁇ l reaction volume containing 2 units of Taq DNA polymerase (Promega Biotech, Madison, Wisconsin) in a 1.5 mM MgCl 2 , 0.2 mM dNTPs, reaction buffer containing 10 pmol of each primer, and 20 ng of template DNA from ps/SAPl . After incubating for 2 min. at 94°, amplification was carried out in a Coy Tempcycler via 35 cycles of (1) 1 min. at 94°; (2) 1 min. at 55°; (1) 1 min. at 72°; followed by 10 min. at 72°; and storage at 4°.
  • a 195 bp band corresponding to the SOE product was then isolated from a 3 % agarose/TBE gel stained with 0.5 ⁇ g/ml EtBr and DNA was isolated using a 0.45 ⁇ m spin filter (Millipore).
  • the isolated DNA was cut with the restriction endonuclease Bam HI and cloned into the Bam HI site of the expression vector pDT-1 described above to generate plasmid pADA/SAP+Ll.
  • the orientation of the inserted DNA was determined by restriction endonuclease digestion and the sequence of the inserted DNA was determined by dideoxy sequencing as described above.
  • This sequence corresponds to that encoding 17 amino acid leader and 43 amino acid ⁇ amyloid peptide portions of the human amyloid precursor protein
  • APP Kang et al, supra, with the exception of a single base substitution in the leader sequence (shown in bold and underlined) which does not affect amino acid composition of the peptide encoded.
  • a map of the ADA sequence, the /3/A4 sequence, and the SV40 polyadenylation sequence as present in pADA/3AP+Ll is shown in Figure 1.
  • the plasmid pADAjSAP+Ll also contains a T7 promoter to allow production of /3/A4 antisense RNA.
  • the plasmid pADAhACT-1 was deposited with the American Type
  • the transgene construct was prepared containing the entire coding region of the human ACT gene inserted the expression vector described by Rauth, et al, supra.
  • the coding sequence of the human ⁇ l-antichymotrypsin cDNA used in the construction of plasmid pADAhACT was prepared by PCR amplification of the full length human ACT DNA insert contained in pHACT as described by Chandra, T. et al. Biochem. 22:5055-5061 (1983).
  • ACT-A 5' ATACCCGGGCAGAGTTGAGAATGGAGA 3' (SEQ ID NO. 9) representing the 5' end of the human ACT cDNA;
  • ACT-B 5' ATACCCGGGTTACTGAGAGCCCCACTG 3' (SEQ ID NO. 10) representing the 3' end of the human ACT cDNA.
  • PCR was carried out in a 500 ⁇ l volume with 10 units of Taq polymerase (Promega Biotech, Madison, WI) in a 1.5 mM MgCl 2 , 0.2 mM dNTPs in reaction buffer containing lOOng of linearized template DNA and 100 pmole each of primers ACT-A and ACT-B.
  • Template DNA was linearized pHACT.
  • Amplification was carried out after incubating for 2 min. at 94°, in a COY Tempcycle via 35 cycles of (1) 1 min at 94°C; (2) 1 min. at 60°C; and (3) 1 min. at 72°C; followed by 10 min. at 72°C and storage at 4°C. Amplification products were analyzed by electrophoresis on 0.8% agarose gels. A 1.3 kb fragment containing the ACT coding sequence was eluted from the gel.
  • the ACT coding sequences obtained above were cloned into the expression vector described in Example 1 wherein the Nco I site surrounding the ATG start codon of the ADA cDNA fragment was destroyed by digestion with SI nuclease so that the first in frame ATG codon after the major start site of transcription was the start site normally used in the human ACT gene.
  • the ACT coding sequence was digested with Sma I to obtain blunt ends to which Bam HI linker were ligated.
  • the Bam HI linkers were used to clone the ACT sequence containing fragment into a unique Bam HI site of the expression vector.
  • the orientation of the insert was determined by restriction digests and the sequence of the inserted DNA was verified by dideoxy sequencing using a kit from U.S.
  • the plasmid pADAhACT- 1 also contains a T7 promoter useful in the production of ACT antisense RNA.
  • Both pADAhACT and pADA/SAP+Ll were purified by centrifugation through CsCl gradients as described in Rauth et al. , supra.
  • a 4.1 kb DNA fragment containing the 5' region of the mouse adenosine deaminase gene, the human ACT cDNA and the SV40 polyadenylation sequence (ACT transgene) was excised from pADAhACT by digestion with the restriction endonucleases Nco I and Hind HI.
  • the 4J kb fragment was separated by electrophoresis in 0.6% low melting point agarose gel, excised from the gel and purified by phenol extraction and ethanol precipitation.
  • the purified 4J kb fragment was then injected into the male pronucleus of single cell embryos from an outbred ICR strain mouse originally of Swiss Webster background (Charles River Laboratories Boston, MA and Jackson Laboratory, Bar Harbor, Maine and National Cancer Institute Fredrickson, Maryland) using the method described by Ross, et al, Proc. Nat'l Acad. Sci. USA 82:5880-5884 (1985). Injected embryos were then implanted into pseudopregnant females as described in Ross et al. , supra and allowed to proceed to term.
  • a 2.9 kb fragment containing the 5' region of the mouse adenosine deaminase gene and the APP leader sequence fused to the 5' end of the 3/A4 coding sequence and the SV40 polyadenylation sequence (j8/A4 transgene) was excised from the plasmid pADA ⁇ AP+Ll using Nco I and Hind HI. The fragment was purified as described above and single cell mouse embryos were injected with the purified 2.9 kb transgene.
  • Genomic DNA was prepared from tail tissue of potential founder mice produced by the method of Example 3 according to the method described in Rauth et al , supra. DNA was then analyzed for the presence of the ACT transgene or the /S/A4 transgene by Southern hybridization. Genomic DNA (20 ⁇ g) was digested to completion with Eco RI and was analyzed by electrophoresis on 0.8% agarose gels/ Gels were blotted and the blots were hybridized with 32 P-labeled probes corresponding to the SV40 polyadenylation sequences. SV40 sequences were used as probes to eliminate the possibility that coding sequence probes might cross-react with endogenous ACT or /S/A4 coding sequences.
  • Blots were then washed at 68°C for 30 minutes in 2x SSC twice, at 68°C for 30 minutes in lx SSC twice and finally for 30 minutes in 0.5x SSC. Blots were then exposed to x-ray film at -70°C before developing.
  • five founder mice were identified by virtue of the presence of a 4J kb band on Southern blots corresponding to ACT transgene DNA. Founder mice were bred to non-transgenic ICR mice and three of the founders (strain numbers 5, 6, and 19) showed transmission of the transgene to their offspring.
  • Figure 3 shows Southern blot analysis DNA from one-month old offspring from strain #19 (lanes 1 and 2), strain #5 (lanes 3-6), and strain #6 (lanes 7-11) and reveals a band of about 4J kb which hybridized to the SV40 polyadenylation sequence probe. Since the ACT construct contains a single Eco RI (see Figure 2) site, the presence of 4.1 kb band indicates that the ACT construct was integrated into the mouse genome as tandem repeats. Lane 12 contains genomic DNA derived from tail tissue of a non-transgenic mouse and does not show the presence of DNA corresponding to the transgene.
  • ACT transgene expression in the brains of each strain of transgenic mice were analyzed by a reverse transcriptase - polymerase chain (RT- PCR) reaction.
  • Transgenic animals carrying the ACT transgene and wild-type animals were sacrificed at 2 months of age, and total brain RNA was prepared as described in Davis et al. eds. Basic Methods in Molecular Biology, Elsevier pp. 130-135 (1986).
  • RT-PCR was performed as described by Kawasaki, E.S. Amplification of RNA, in PCR Protocols, A Guide to Methods and Applications. Innis, M.A. et al. eds., Academic Press Inc. , pp. 21-28 (1990).
  • RT-PCR was carried out by reverse transcribing 1 ⁇ g of total brain RNA using 200 units of Moloney Murine Leukemia Virus reverse transcriptase (Gibco-BRL, Gaithersberg Maryland) and 100 pmoles of random hexamers (primers) (Pharmacia-LKB, Piscataway NJ) in a total reaction volume of 20 ⁇ l according to the manufacturer's instruction.
  • Transgene specific primers were chosen so that a 340 base pair fragment from the 3' end of the ACT transcript would be amplified.
  • the primer sequences used are as follows:
  • rPCRhACTl-5' 5' CACCAGCAAGGCTGACCTGT (SEQ ID NO. 11); rPCRhACTl-3': 5' GTTGTGGTTTGTCCAAACTC (SEQ ID NO. 12).
  • PCR amplification was carried out in a 50 ⁇ l reaction volume using 2 units of Taq Polymerase (Promega Biotech) in 1.5 mM MgCl 2 , 0.2 mM dNTPs, 4 ⁇ l of the reverse transcription reaction and 10 pmoles of each transgene specific primer. All reaction components were assembled and, after a 2 min. incubation at 94°C, amplification was carried out in a COY Thermocycle at the following settings: 1 min. at 94°C; (2) 1 min. at 60°C; and (3) 1 min. at 72°C; followed by 10 min. at 72°C and storage at 4°C. Half of each reaction was then analyzed by electrophoresis on a 2% agarose gel and staining with ethidium bromide (0.5 ⁇ g/ml).
  • Figure 5 shows the results of the RT-PCR analysis.
  • RNA derived from the brains of transgenic mouse strain #19 (Lanes 1 and 2), strain #5 (lanes 3 and 4), strain #6 (lanes 6 and 7), and wild-type mice lacking the ACT transgene (lanes 8 and 9) were analyzed as described above.
  • Lanes 1, 3, 5 and 9 represent reactions which were reverse transcribed prior to amplification
  • lanes 2, 4, 5 and 9 represent control reactions which were performed without reverse transcription prior to amplification.
  • Additional positive and negative amplification controls consist of amplification reactions performed using either tail DNA derived from one of the transgenic mice (lane 9) or in the absence of any nucleic acid template (lane 10).
  • the expected PCR product generated if transgene specific RNA was present was 340 bp in size.
  • ACT transgene specific RNA showed expression in the brain as revealed by the presence of a 340 bp band (lanes 1, 3, 5) on agarose gel electrophoresis which was also seen when DNA from a transgenic mouse containing the ACT transgene was used as a template for PCR (lane 9).
  • the antisense probe used corresponds to a 590 bp fragment from the 5' end of the human ACT gene.
  • Hybridization was carried out in hybridization buffer containing 50% formamide, 10 mM dithiothreitol (DTT), 2 x SSC, 1 mg/ml yeast tRNA, 1 mg/ml bovine serum albumin (BSA), 1 mg/ml sonicated salmon sperm DNA.
  • Hybridization was carried out on a slide warmer at 55°C 1 mg/ml with 15 ⁇ l hybridization buffer per slide under glass cover slips (Corning, N.Y.) sealed with rubber cement to prevent evaporation.
  • Figure 6 shows the results of in situ hybridization analyses. High level expression of the ACT transgene has seen throughout the CNS (by virtue of hybridization of the ACT specific antisense probes shown by the presence of silver grains) including cortex (C), hippocampus, dentate gyrus (O), basal ganglia, thalamus (T), hypothalamus (H), and cerebellum (Cb).
  • Figure 6, panel A represents a coronal section from a transgenic mouse (strain 5) illustrating ACT expression in the hippocampus and surrounding cortex.
  • Figure 6, panel B shows a coronal section from a wild-type mouse hybridized with the same probe which shows no hybridization.
  • FIG. 6 panel C represents a sagittal section from a transgenic mouse (strain 5) showing ACT expression throughout the brain.
  • Figure 6, panel D represents a sagittal section from a wild-type mouse hybridized to the same ACT transgene which shows no hybridization.
  • the granule cell layer of the hippocampus and the dentate gyms were the most intensely labeled.
  • Layer 1 of the cerebral cortex, which contains relatively few neurons had the lowest hybridization signal in the cortex, while other more neuron-rich regions showed more intense labeling.
  • the brains of human patients with Alzheimer's disease show characteristic lesions including diffuse amyloid plaques, neurofibrillary tangles, senile neuritic plaques and others.
  • ACT transgene expression of the ACT transgene in transgenic mice correlated with the appearance of any of the lesions described above
  • eight-month-old transgenic offspring from each of the three strains (5, 6, and 19), as well as control mice of varying ages were analyzed using two different methods.
  • Brains were prepared for analysis by perfusing mice systemically with 10% formalin. Brains were removed and post-fixed in 40% formalin for 3-7 days at 4°C, and cryoprotected by immersion in 30% sucrose until tissue sank to the bottom of the container. Brains were then frozen on powdered dry ice and sectioned at 50 ⁇ m intervals on a cryostat. Series of parasagittal sections were impregnated with silver according to the method of Campbell et al , Soc. Neurosci. Abst. 13:678 (1987) which has previously been shown to be both highly sensitive and specific in the staining of the /S/A4 amyloid lesions found in the brain of AD patients.
  • Figure 7 shows photomicrographs of parasagittal sections of the brain of a transgenic mouse expressing the ACT transgene (panels A-E), and an age-matched non-transgenic ICR control mouse (panel F).
  • Plaque-like lesions morphologically indistinguishable from the diffuse amyloid plaques of AD patients are seen in virtually every region of the transgenic mouse brain including the hippocampus (arrowheads in Panel A, C and E), cerebral neocortex (arrows in Panel D), midbrain (arrowheads in Panel B), basal telencephalic nuclei ( a.k.a basal ganglia) diencephalon, cerebellum, pons, medulla and spinal cord.
  • Panel E shows a higher power view of the hippocampal region from the section shown in Panel C and demonstrates the plaque-like lesions in the hippocampus (arrows) and adjacent neocortex.
  • Panel F illustrates the absence of such lesions in age matched non-transgenic ICR mouse brain. All the brains from transgenic offspring exhibited widespread argyrophilic lesions from 10 to about 250 ⁇ m in diameter (Figure 7). These plaque-like lesions were morphologically indistinguishable from the diffuse amyloid plaques found in the brain of AD patients. Tagliavini, et al, Neuropathol. Exptl. Neurol 47:332; Yamaguchi, et al, Acta Neuropathol. 76:541-544 (1988); Joachim, et al, Am. J. Pathol 135:309-319 (1989); Bugiani, etal, Neurosci. Lett.
  • the lesions were situated in virtually every region of the mouse brains, including the hippocampus, diencephalon, cerebral neocortex, basal telencephalic nuclei (a.k.a. "basal ganglia"), midbrain, cerebellum, pons, medulla and spinal cord.
  • mice were sacrificed and fixed in formalin as described in Example 7. Brains were then frozen and series of 50 ⁇ m thick parasagittal sections were obtained on a cryostat. Brain sections were incubated in 1:500 - 1:2000 dilutions of rabbit anti- ⁇ l-antichymotrypsin in phosphate buffered saline - sodium azide. (Zymed Laboratories, Inc. So. San Francisco, CA 94080). Brains were then processed according to the avidin-biotin-peroxidase method described by Kuljis, R. et al , Neurosci. Lett. 106:49-54 (1989). Human tissues showing histologically confirmed AD were obtained at the time of autopsy and were processed for labeling for ACT following the same method used for mice.
  • FIG 8 illustrates the results of this analysis.
  • panels A-D illustrate the presence of immunolabeled ACT containing plaques in the brains of transgenic mice.
  • the brains of human patients with AD also show the presence of immunolabeled ACT (Panels E and F).
  • sections from transgenic mice stained with rabbit antihuman anti- ⁇ l-antichymotrypsin displayed plaque-like or cloud-like aggregations of ACT immunoreactive material within the plaques. These aggregations appeared in the parenchyma proper (Panels A and B), in the subpial region (from the submolecular layer of the cerebellum) (Panel C) and in the perivascular spaces (Panel D).
  • Example 7 Additional studies were conducted to determine whether the plaque-like lesions described in Example 7 contained the APP-derived amyloid protein. Series of sections adjacent to those taken in Example 7 were immunolabeled with a panel of antibodies including a polyclonal antiserum raised against the /S/A4 fragment of the human amyloid protein as described by Masters C.L. et al , EMBO J. 41:2257-2763 (1985) (the "Masters antiserum”), a polyclonal antiserum raised against synthetic ⁇ - amyloid (Boehringer Mannheim, Indianapolis, Indiana Cat. No.
  • mice were fixed and prepared for histology as described above and tissues were treated with antibodies in dilutions of 1:500-1:2000 after pretreatment of the tissue with 70% formic acid. Tissues were then stained using the avidin-biotin- peroxidase method as described by Kuljis et al, supra. The best results were obtained in material intensified according to the protocol of Gallyas et al, J. Histochem. and Cytochem. 30:183, (1982) after immunolabeling. Tissue from transgenic and age-matched control mice and from patients with confirmed AD was processed under identical conditions.
  • Figure 9 A-C illustrate 8/A4 immunopositive deposits in transgenic mouse brain immunolabeled with the Masters antiserum.
  • Figure 9 A depicts lesions in the interface between the pontine tegmentum and base.
  • Figure 9B depicts lesions in the dorsal pontine tegmentum.
  • Figure 9C depicts lesions in the molecular layers of the cerebellum.
  • PCL denotes the Puririnje cell layer and the arrows delineate the borders of a large /3/A4 deposit.
  • plaques were 8- 55 ⁇ m in diameter and displayed irregular contours. They contained a variable admixture of 8-12 ⁇ M clumps, puncta, and a hazy amorphous background that clearly distinguished them from surrounding unlabeled tissue. These lesions were situated in the same regions in which agyrophilic plaques were seen in adjacent sections as described in Example 7. No plaque-like lesions or deposits were observed with these methods in any of the five control non-transgenic ICR mice (ranging in age from 4-17 months). Furthermore, control mice did not show amyloid type deposits of any type, including the type seen occurring spontaneously in C57/BL6 as reported by Jucker et al, supra. Thus, the amyloid-containing plaques observed in the transgenic animals described herein are attributable to the presence of the ACT transgene.
  • the transgenic animals of the present invention may be used to test potential therapeutic interventions for the treatment and prevention of AD disease.
  • Potential interventions range from but are not limited to traditional types of pharmacologic intervention (e.g., drugs) to molecular therapeutic interventions including the utilization of antisense RNA, ribozymes, tagged-triplex DNA/RNA molecules, small peptides derived from dominant negative mutations, peptide nucleic acid molecules (PNA), peptides interfere with the biochemical process of plaque deposition, antibodies directed to plaque-producing proteins and others, all of which are well known in the art. These treatments may be administered after the onset of plaque-formation or may be used in a prophylactic capacity.
  • pharmacologic intervention e.g., drugs
  • molecular therapeutic interventions including the utilization of antisense RNA, ribozymes, tagged-triplex DNA/RNA molecules, small peptides derived from dominant negative mutations, peptide nucleic acid molecules (PNA), peptides interfere with the biochemical process of plaque de
  • mice may be sacrificed at various times after intervention and their brains examined for the presence of diffuse amyloid plaques, neurofibrillary tangles and other histologic signs of Alzheimer's disease using the standard histologic and immunohistologic techniques described above, which are widely known in the art.
  • the brains from these animals may also be examined for the presence of ⁇ l-antichymotrypsin-specific or ⁇ /AA- specific mRNAs using RT-PCR, Northern blotting RNase protection assays or by in situ hybridization.
  • Those therapeutic interventions showing an effect on the expression of transgenes and/or on the process of plaque formation would then merit more intensive investigation.
  • the present invention is also directed to transgenic animal model systems comprising non-human mammals other than mice.
  • Such animal models may differ from mice in their natural aging processes and rate of aging and will allow a more detailed investigation of the role of aging in the onset of plaque formation in transgenic animals.
  • Transgenic animals including transgenic rabbits, transgenic pigs, transgenic rats, and other transgenic animals have been described. (See for example, Hammer, et al, Nature 315:680-683 [1985]).
  • Transgene constructs useful in other transgenic animal model systems for AD would include regulatory sequences capable of directing expression of a coding sequence in the neurons of transgenic animals as well as other sequences such as leader sequences which may be important to protein secretion process.
  • the coding sequences useful in such constructs include the coding sequence for ACT, ⁇ lAA peptide encoding sequences and other coding sequences whose expression can result in the development of the characteristic histopathologic signs of AD.
  • animals of the present invention may be taught certain tasks such as the Morris water maze task which measures spatial learning in animals. As the disease progresses in the animal as evidenced by the increasing abundance of plaques and other pathophysiologic features of AD, the animals may be retested for their ability to perform the same tasks or for their ability to learn new tasks. Other methods for testing learning (e.g. avoidance testing) are within the skill of the ordinary person in the art of animal psychology, pharmacology, neurology and physiology.
  • Proposed environmental causes of AD include head trauma, viral infection, aluminum toxicity and others. Gautrin et al, Can. J. Neurol Sci. 16:375- 387 (1989). One common aspect among these otherwise highly diverse environmental factors is their potential for causing localized inflammation within the brain. A major feature of the inflammatory response is the rapidly increased production of a set of "acute phase response" proteins among which ACT is one of the earliest and most prominent. Travis, et al , Ann. Rev. Biochem. 52:655-709 (1983). Other acute phase response proteins which have been postulated to play a role in the formation of amyloid in the brain are the cytokines IL-l/IL-6. Vandenabeele et al , Immunol. Today 12:217-219 (1991).
  • transgenic animals of the present invention may be used to test whether or not the aforementioned environmental insults to the brain can in fact influence the rate of formation or the extent of AD-like pathology.
  • animals of the present invention may be infected with viruses suspected of playing a role in the etiology of AD after which expression of the transgene or plaque formation may be monitored.
  • animals of the present invention may be used to investigate the effects of head trauma, and environmental toxins including metals and organic chemicals, on the progression of the neurologic and pathologic signs of AD.
  • the present invention is also directed to cell culture systems which express genes which play a role in the development of AD.
  • Cell cultures derived from the transgenic animals may be used to test agents which may influence the expression of genes which play a role in the development of AD.
  • Cell cultures derived from the animals of the present invention may also be used as test systems for agents or interventions which may alter the translation of mRNAs such as ACT mRNA or ⁇ lAA mRNA or which may alter the post-translational modification of proteins such as ⁇ lAA peptide or ACT.
  • the preparation of cell cultures from animals can be accomplished using standard techniques known to those of ordinary skill in the art of cell culture.
  • Cell lines useful in the study of AD may also be prepared by transfecting cells in culture with transgenes of the present invention. Transfection may be accomplished using well-known techniques such as electroporation, calcium phosphate co-precipitation, retrovirus vector mediated gene transfer, microinjection or other techniques known in the art.
  • Transfection may be carried out by cotransfecting the transgene of interest with a selectable marker such as the bacterial neo* marker which confers resistance to the antibiotic G418 on cells expressing the neo r .
  • a selectable marker such as the bacterial neo* marker which confers resistance to the antibiotic G418 on cells expressing the neo r .
  • a significant percentage of the cells expressing the neo r gene will also express the cotransfected transgene.
  • transformed cells surviving selection in G418 may then be assayed for expression of the transgene by any of a wide variety of techniques known to those of ordinary skill in the art.
  • Transfected cells may also be prepared as described above without cotransfection with a selectable marker. In that case cells may be transfected with a transgene and assayed for expression of the transgene from about 24 to about 72 hours after transfection without any intervening selection.
  • pADAhACT was transfected into rat cells in culture. More specifically, subconfluent monolayers of rat pheochromocytoma cells (PC-12 cells) were cotransfected with the plasmid pADAhACT and a pTZ19U based plasmid containing (in 5' to 3' orientation) the minimal ADA promoter (Rauth et al.
  • ORGANISM Human ⁇ l-antichymotrypsin
  • ORGANISM Human ⁇ l-antichymotrypsin
  • ORGANISM Human ⁇ l-antichymotrypsin
  • ORGANISM Human ⁇ l-antichymotrypsin

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Abstract

L'invention concerne des systèmes de modèles animaux transgéniques de la maladie d'Alzheimer. Plus précisément, l'invention porte sur des mammifères transgéniques non humains surexprimant la α-1-antichymotrypsine ou le peptide βA4 dans le cerveau, principalement dans les neurones, ainsi que sur des sytèmes d'essais utiles pour des interventions thérapeutiques et prophylactiques, sur la maladie d'Alzheimer. Par ailleurs, des lignées cellulaires provenant des animaux transgéniques selon l'invention sont également décrites.
PCT/US1994/004026 1993-04-19 1994-04-12 Modeles animaux transgeniques pour la maladie d'alzheimer WO1994024266A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5912410A (en) * 1990-06-15 1999-06-15 Scios Inc. Transgenic non-human mice displaying the amyloid-forming pathology of alzheimer's disease
WO2002005634A2 (fr) * 2000-07-13 2002-01-24 University Of South Florida Animal transgenique et methodes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Volume 22, No. 22, issued 25 October 1983, T. CHANDRA et al., "Sequence Homology Between Human Alpha1-Antichymotrypsin, Alpha1-Antitrypsin and Antithrombin III", pages 5055-5060. *
NATURE, Volume 341, issued 12 October 1989, NOSTRAND et al., "Protease Nexin-II, a Potent Antichymotrypsin, Shows Identitiy to Amyloid Beta-Protein Precursor", pages 546-549. *
NATURE, Volume 352, issued 18 July 1991, D. QUON et al., "Formation of Beta-Amyloid Protein Deposits in Brains of Transgenic Mice", pages 239-41. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5912410A (en) * 1990-06-15 1999-06-15 Scios Inc. Transgenic non-human mice displaying the amyloid-forming pathology of alzheimer's disease
WO2002005634A2 (fr) * 2000-07-13 2002-01-24 University Of South Florida Animal transgenique et methodes
WO2002005634A3 (fr) * 2000-07-13 2003-09-25 Univ South Florida Animal transgenique et methodes
US6781029B2 (en) 2000-07-13 2004-08-24 University Of South Florida Transgenic animal and methods

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