EP0724628A1 - Modele animal transgenique pour le traitement de troubles cognitifs - Google Patents

Modele animal transgenique pour le traitement de troubles cognitifs

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Publication number
EP0724628A1
EP0724628A1 EP94922619A EP94922619A EP0724628A1 EP 0724628 A1 EP0724628 A1 EP 0724628A1 EP 94922619 A EP94922619 A EP 94922619A EP 94922619 A EP94922619 A EP 94922619A EP 0724628 A1 EP0724628 A1 EP 0724628A1
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European Patent Office
Prior art keywords
animal
dna
human
gene
mouse
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP94922619A
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German (de)
English (en)
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EP0724628A4 (fr
Inventor
Howard Y. Chen
Kathryn J. Hofmann
Leonardus H.T. Van Der Ploeg
Alan R. Shaw
Myrna E. Trumbauer
Hui Zheng
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Merck and Co Inc
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Merck and Co Inc
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Publication of EP0724628A1 publication Critical patent/EP0724628A1/fr
Publication of EP0724628A4 publication Critical patent/EP0724628A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • 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
    • 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
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0368—Animal model for inflammation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

  • the present invention relates to the expression of human interleukin- l ⁇ in brain tissues of transgenic mice.
  • AD Alzheimer's Disease
  • AD is a neurological disorder affecting the population over 65 years of age. Incidence of the disease increases from less than 1 % at age 60-65, to 5% at age 75, to as high as 47% at age 85. As a result 60% to 80% of all cases of dementia in persons over age 65 are caused by AD. Afflicted individuals exhibit impaired cognitive function and memory. AD pursues a rapid course leading to mental and physical incapacity before death occurs, generally five to ten years after onset of the symptoms. Currently there is no effective treatment for AD and the biochemical basis of the disease remains obscure.
  • AD presents itself in late life without clear markers that explain the onset of disease. Viral, environmental and immunological factors have been implicated in the development of AD. In contrast to the late-life onset of AD (sporadic disease), familial AD (FAD) is characterized by a more rapid progression of the disease and an early onset in life (as early as 35 years of age).
  • FAD familial AD
  • the dementia associated with AD correlates to the remarkable atrophy of AD brain, which is widespread throughout the brain, involving the neocortex, hippocampus, parahippocampal structures including the entorhinal cortex, olfactory cortex and bulb, the cholinergic forebrain basal nucleus, the dorsal serotonergic nuclei and the locus ceruleus noradregergic nuclei.
  • AD Amyloid Precursor Protein
  • APP Amyloid Precursor Protein
  • indirect biochemical evidence hints at the involvement of the APP, which is synthesized in neurons.
  • abnormal structures can be identified associated with degenerating neurons. These structures which contain a proteolytic cleavage product of APP, the ⁇ A4 peptide, are referred to as neuritic plaques (NP) and are located within the neocortex and the hippocampus.
  • NP contain several proteins. The most abundant component is the -42 amino acid ⁇ A4 peptide which is proteolytically cleaved from the APP. The presence of the ⁇ A4 is significant since several reports have outlined a potential neurotoxic effect of this peptide in vitro.
  • APP is a transmembrane protein expressed in several tissues. Different spliced forms of the APP mRNA give rise to several proteins. The major forms of APP are known as APP 751, APP 770 and APP 695. APP 751 and 770 encode a 56 amino acid insert known as the kunitz protease inhibitor domain while APP 695 lacks the kunitz domain. APP processing has been extensively studied.
  • the APP can be cleaved at amino acid position 669 (secretase cleavage site at amino acid 16 of the ⁇ A4, thus preventing ⁇ A4 production) and produces a large secreted peptide known as nexin II (encoding the kunitz protease inhibitor domain, which can inhibit secretase cleavage). Cleavage at amino acids 653 and 695 can lead to release of the ⁇ A4 peptide. This cleavage may occur in the endolysosomal compartment.
  • the mutations in the APP outlined above are believed to affect the efficiency of APP processing thus increasing the susceptibility to abnormal degradation of the APP protein.
  • Interleukin- 1 represents a family of three cytokines, IL-lcc, IL-l ⁇ and IL-lra.
  • EL- lot and IL-l ⁇ are produced principally by macrophages and are commonly acknowledged as major drivers of chronic and acute inflammation.
  • IL-l ⁇ and L -l ⁇ are only distantly related gene products, they share a complete spectrum of properties (size, 3-D shape, biological activity, cell source).
  • IL-1 receptor antagonist (EL- Ira) is an IL-like molecule which binds and blocks the type I IL-1 receptor, thus inhibiting IL-l ⁇ and IL-l ⁇ action.
  • macrophage infiltrates are found around degenerating neurons in AD.
  • macrophages are the first cells recruited to the area of cell degeneration.
  • Fourth, IL-1 mRNA and activity are found in AD brain tissue.
  • APP synthesis is inducible by cytokines, specifically by IL-6.
  • head injury and subsequent inflammation can lead to the early development of AD. Given the abundance of APP production in AD it has been proposed that an D -l/IL-6 mediated acute phase response may be responsible for amyloidogenesis during AD.
  • mice of the present invention are useful in the identification of compounds that affect APP production and compounds that affect IL-l ⁇ mediated neuronal dysfunction.
  • a transgenic mouse expressing human interleukin- l ⁇ is provided.
  • the transgenic mouse of the invention may be used in the study of Alzheimer's Disease and disorders involving the central nervous system. DETAILED DESCRIPTION OF THE INVENTION
  • animal is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
  • a "transgenic animal” is any animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by micromjection or infection with recombinant virus. This introduced DNA molecule may be integrated within a chromosome, or it may be extra-chromosomally replicating DNA.
  • the term “germ cell-line transgenic animal” refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information then they, too, are transgenic animals.
  • the information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or genetic information already possessed by the recipient.
  • the introduced gene may be differently expressed than the native gene.
  • the genes may be obtained by isolating them from genomic sources, by preparation of cDNAs from isolated mRNA templates, by directed synthesis, or by some combination thereof.
  • the structural gene must be coupled to a promoter in a functional manner.
  • Promoter/regulatory sequences may be used to increase, decrease, regulate or designate to certain tissues or to certain stages of development the expression of a gene.
  • the promoter need not be a naturally occurring promoter.
  • the human Thy-1 (hThy-1) promoter is used.
  • the hThy-1 promoter preferentially allows expression of the gene of interest, here the human interleukin- l ⁇ predominantly within the brain (Gordon, J., et al., CeH 50:445-452, (1987)).
  • transgenic non-human animals are produced by introducing "transgenes" into the germline of the non- human animal.
  • the methods enabling the introduction of DNA into cells are generally available and well-known in the art; however, the generation of a particular type of transgenic animal requires experimentation.
  • Embryonal target cells at various stages of development can be used to introduce transgenes. Different methods of introducing transgenes are used depending on the stage of development of the embryonal target cell. Generally, the zygote is the best target for microii jection. In the mouse, the male pronucleus reaches the size of approximately 20 ⁇ m in diameter which allows reproducible injection of 1-2 pL of DNA solution.
  • zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster, et aL, (1985) Proc. Natl. Acad. Sci. USA 82, 4438-4442). Consequently, nearly all cells of the transgenic non-human animal will carry the incorporated transgene. Generally, this will also result in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Micromjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.
  • Retroviral infection can also be used to introduce a transgene into non-human animal.
  • the developing non-human embryo can be cultured jn vitro to the blastocyst stage.
  • the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad. Sci. USA 73, 1260-1264).
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al., (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
  • the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et aL, (1985) Proc. Natl. Acad. Sci. USA 82, 6927-6931; Van der Putten et aL, (1985) Proc. Natl. Acad. Sci. USA 82, 6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra: Stewart et aL, (1987) EMBO J. 6: 383-388). Alternatively, infection can be performed at a later stage.
  • Virus or virus-producing cells can be injected into the blastocoele (Jahner et aL, (1982) Nature 298: 623-628). Most of the founder animals will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder animal may contain retroviral insertions of the transgene at a variety of positions in the genome; these generally segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (Jahner et aL, (1982) supra).
  • ES cells are obtained from pre-implantation embryos cultured in vitro (Evans. M. J., et aL, (1981) Nature 292, 154- 156; Bradley, A., et aL, (1984) Nature 309, 255-258; Gossler, et aL, (1986) Proc. Natl. Acad. Sci. USA 83, 9065-9069; and Robertson, et aL, (1986) Nature 322, 445-448).
  • Transgenes can be efficiently introduced into ES cells by DNA transfection or by retrovirus-mediated transduction.
  • the resulting transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
  • the ES cells colonize the embryo and contribute to the germ line of the resulting chimeric animal (For review see Jaenisch, R. (1988) Science 240, 1468- 1474).
  • PCR DNA polymerase chain reaction
  • PAGE polyacrylamide gel electrophoresis
  • Western Blots Western Blots to detect IL-l ⁇ protein and the appearance of pathology associated with Alzheimer's Disease.
  • a "transgene” is a DNA sequence introduced into the germline of a non-human animal by way of human intervention such as by way of the methods described below.
  • the transgenes of the invention include DNA sequences which are capable of suppressing cognate endogenous alleles. Attempts to express human interleukin l ⁇ in transgenic animals have met with minimal success. There may be several reasons for this. First, EL-l ⁇ is toxic at relatively low concentrations. Second, IL-l ⁇ has been implicated in embryonic development and parturition. Third, IL-l ⁇ is produced as a precursor which is processed for release from specific types of cells (e.g., macrophages) by a specific mechanism.
  • IL-l ⁇ a targetted expression system was developed.
  • the gene encoding human interleukin- l ⁇ was placed downstream of the human Thy-1 promoter.
  • the hThy-1 promoter specifically targets mouse brain tissue.
  • Transgenic mice carrying the artificial Thyl-IL-l ⁇ gene express hIL-l ⁇ in brain tissue.
  • the overexpression of IL-l ⁇ may increase the synthesis and processing of amyloid precursor protein.
  • a potential therapeutic compound for Alzheimer's Disease may be detected by measuring its capacity to block the production of amyloid protein precursor (APP) in these transgenic mice.
  • APP amyloid protein precursor
  • Such compounds will be formulated in accord with known methods to produce pharmaceutically acceptable compositions. Such compositions may be administered to patients in a variety of standard ways.
  • plasmid p!2849-57-2 pBSHTl contains a 8kb EcoRI fragment of human Thy-1 gene (hThy-1) in pBSV (Van Rijs et aL, Proc. Natl. Acad. Sci. USA 82:5832-5835, 1985).
  • a PCR amplication was carried out using pHZ021a as a template and oligos T7 (in pTZ18u backbone) and oHZ002 (at the ATG initiation codon) as primers to generate a 1.3 kb product.
  • the sequence of the T7 primer is 5'-TAA TAC GAC TCA CTA TAG GG-3' (SEQ ID NO: 1).
  • the oHZ002 nucleotides destroyed the ATG codon and introduced a polylinker cloning site in the PCR product, as follows: 5'-ACGTCGACTCTAGAAGATCTTCGACTCGAGATCGATGGT ACCCGGGCAGGTTCAAGCTTCTGGGATCTCAGTC-3' 5'-TCATGGTTCTGGGATCTCAGTC-3' Wild-type
  • a Bgi ⁇ linker d(CAGATCTG) was inserted at the Smal site upstream of the SV40 small T intron of pSV2neo (pHZ023). (Southern, P.J. & Berg, P.J. Mol. Appl. Genet. 1:327, 1982.).
  • the 1.0 kb S V40 small T intron and polyA was isolated by Bgi ⁇ and BamHI digestion of pHZ023 and ligated into the Bglll digested pHZ022.
  • the resulting plasmid, pHZ024 contains two regulatory elements: the human Thy-1 promoter for brain-specific expression and the SV40 small T intron and polyA sequence for proper expression of the transgene.
  • the ATG initiation codon of the hThy-1 gene was mutated and a polylinker cloning site was introduced downstream of the hThy-1 promoter; this permitted insertion of genes to be expressed in transgenic mice.
  • Plasmid pHZ024 was digested with Cla I and BglU which cut within the polylinker located between the hThy-1 promoter and the SV40 small T and poly A sequences.
  • the 6.3- kb Cla I- Bglll vector fragment was gel-isolated.
  • Two synthetic oligodeoxynucleotides were annealed to form a 86-bp linker with the following structure:
  • the 86-base pair (bp) linker contains a Clal sticky end, 5- bps of untranslated leader sequence from the hThy-1 gene (Seki, et aL, PNAS 82, 6657-6661, 1985), ATG codon (underlined), 17-bp of rat IL- 1 receptor antagonist (IL-lra) signal peptide sequence and the remaining 58-bp of the IL-lra signal peptide sequence from mouse.
  • the chimeric rat/mouse IL-lra signal peptide sequence was used because the first 17-bp of the mouse sequence was unknown.
  • the gene encoding the mature form of the hlL-l ⁇ was constructed using PCR.
  • the template DNA for PCR was plasmid pGEM-Blue/human IL-l ⁇ (gift of Andrew Howard, Merck Research Laboratories, Rahway, NJ). This plasmid contains the cDNA encoding the mature form of hEL-l ⁇ .
  • pGEM-Blue/human IL-l ⁇ was constructed by subcloning the EcoRI- AccI IL-l ⁇ DNA fragment from pKK223-3/hIL-l ⁇ (Tocci, M.J., et aL, Journal of Immunology 138, 1109-1114, 1987) into the EcoRI-AccI digested pGEM-Blue vector (Promega, Inc.).
  • PCR-primers had the following sequences: 5'-GCACCTGTACGATCACTGAACTGC-3' (SEQ ID NO: 4) and 5 * -GAAGATCTAGGAAGACACAAATTGCATGGTGAAG-3' (SEQ ED NO: 5).
  • the 0.5-kb h L-l ⁇ gene fragment was made flush-ended with T4 DNA polymerase, digested with Bglll (which cuts after the stop codon), gel-isolated and phosphorylated with T4 polynucleotide kinase.
  • the 6.3-kb Clal-Bgi ⁇ pHZ024 vector fragment was ligated with the 86-bp IL-lra Clal- blunt linker and the 0.5-kb blunt- Bgi ⁇ mature hlL-l ⁇ gene in a three-way ligation.
  • the resulting plasmid pi 2849-57-2 was sequenced using dideoxynucleotide sequencing.
  • a large- scale CsCl plasmid prep of pi 2849-57-2 was prepared.
  • the plasmid was digested with EcoRI and Xbal and electrophoresed through a 1 % low melting point agarose gel containing 10 ng/ml ethidium bromide.
  • the DNA was visualized using minimal exposure to short-wave UV light and the 5.1-kb DNA band was excised, melted at 65-70°C, phenol/chloroform extracted 3X, chloroform extracted IX and ethanol precipitated in 0.2 M NaCl.
  • DNA was resuspended in 10 mM Tris, 0.25 mM EDTA, pH 7.5 and filtered through a pre-rinsed 0.2 ⁇ m cellulose acetate filter.
  • the 5.1- kb, linear DNA fragment 12849-112-2 containing the hThy-1 promoter, rat/mouse IL-lra signal sequence, mature hIL-l ⁇ gene and SV40 intron and poly A was subsequently used for micromjection.
  • the pi 2849-57-2 DNA construct of Example 1 containing the human IL-l ⁇ driven by the human Thy-1 (hThy-1) promoter was microinjected into the pronucleus of one-cell fertilized mouse embryos obtained from superovulated B6SJL females.
  • the optimal concentration of the DNA used for micromjection was the LD50 value derived empirically from toxicity test experiments using several dilutions of the gene construct microinjected into mouse embryos and was 7.5 x 10 ⁇ 9 ⁇ g.
  • the embryos injected with 7.5 x 10 ⁇ 9 ⁇ g DNA were then surgically reimplanted into the oviducts of pseudopregnant recipient mice and allowed to develop to term.
  • tail samples were taken by clipping off approximately 1 cm of tail for DNA dot blot assay to determine the presence of the transgene. Young pups were closely observed daily starting from PN1 (postnatal day 1) for pathological symptoms. Necropsies and/or biopsies were performed to collect tissue specimens for histological and for expression studies.
  • Example 2 The pups of Example 2 derived from microinjected eggs are weaned at about 4 weeks of age. At that time a small segment (about 1 cm long) was removed from the distal end of the tail and used for DNA analysis. Genomic DNA was extracted from the tail samples and applied to Gene Screen Plus® membrane filter using a dot blot apparatus. The filter was then hybridized with a probe containing SV40 sequence which is present in the 3' area of the transgene. Since the endogenous mouse DNA does not contain the SV40 sequence, this probe is specific for the transgene and can be used to detect as little as 0.1 copies of the transgene in the mouse genome. Transgenic founders identified by DNA dot blot procedure are bred to produce progeny for further studies.
  • RNA-PCR RNA-polymerase chain reaction
  • the oligonucleotides are designed to complement DNA sequences located within exons. Each oligonucleotide pair is separated by one or more introns in order to distinguish between genomic DNA and mRNA.
  • RNA-PCR reaction One ⁇ g of total RNA (determined by absorbance at 260 nm) is used for each RNA-PCR reaction as described (GeneAmp® RNA PCR Kit, product #N808-0017; Perkin Elmer Cetus, Corp.). The PCR is performed for 35 cycles with the following parameters per cycle: 95°C for 1 min., 48°C for 2 min., 72°C for 2 min.
  • DNA bands of the appropriate sizes are visualized on a 1.2% agarose gel containing 0.5 ⁇ g/ml ethidium bromide.
  • the relative mRNA levels (as determined by RNA-PCR) for the tissues of transgenic animals and a control non-transgenic animal are determined.
  • Human IL-l ⁇ mRNA can also be detected by an RNase protection assay. Brain and other tissues are obtained from transgenic mice for isolation of mRNA. A 32p_ ⁇ a beled antisense RNA is used to hydridize hIL-l ⁇ mRNA in a solution hybridization reaction. The resulting double-stranded molecule is resistant to RNase digestion while unhybridized RNA will be digested by RNase treatment. The protected band can be visualized by autoradiography after separation on a polyacrylamide gel.
  • tissue sections are prepared from frozen brain and hybridized with a labeled oligonucleotide probe specific for hlL-l ⁇ mRNA followed by autoradiography.
  • ELISA Enzyme Linked Immunoabsorbant Assay
  • transgenic mice are necropsied to obtain brain and other tissues. Tissue samples are typically fixed in 10% formalin in phosphate buffered saline. Fixed tissues are sectioned mounted on glass slides and read.
  • the transgenic animals of the invention may be used as a source of cells for cell culture.
  • Brain tissues of transgenic mice are analyzed for the presence of human interleukin- l ⁇ , by directly analyzing DNA or RNA or by assaying brain tissue for the protein expressed by the gene.
  • Cells of brain tissues carrying the gene may be cultured using standard culture techniques that are well-known in the art.
  • the animals of the present invention may be used to test compounds for the ability to interfere the expression of human interleukin- l ⁇ or amyloid precursor protein (APP).
  • An animal is treated with the compound, in parallel with an untreated control animal.
  • a comparatively lower level of IL-l ⁇ or APP in the treated animal is an indication of inhibitory activity of the test compound.
  • MOLECULE TYPE cDNA
  • MOLECULE TYPE cDNA

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Abstract

Souris transgéniques dotées du gène de l'interleukine humaine 1β qui peuvent être utilisées pour évaluer les effets de composés agissant sur la maladie d'Alzheimer et d'autres troubles cognitifs.
EP94922619A 1993-07-22 1994-07-19 Modele animal transgenique pour le traitement de troubles cognitifs Withdrawn EP0724628A4 (fr)

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Application Number Priority Date Filing Date Title
US9694493A 1993-07-22 1993-07-22
US96944 1993-07-22
PCT/US1994/008110 WO1995003397A1 (fr) 1993-07-22 1994-07-19 Modele animal transgenique pour le traitement de troubles cognitifs

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EP0724628A1 true EP0724628A1 (fr) 1996-08-07
EP0724628A4 EP0724628A4 (fr) 1997-07-02

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EP (1) EP0724628A4 (fr)
JP (1) JPH09500533A (fr)
CA (1) CA2167581A1 (fr)
WO (1) WO1995003397A1 (fr)

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JPH10507637A (ja) * 1994-10-20 1998-07-28 メルク エンド カンパニー インコーポレーテッド インターロイキン−１β欠損トランスジェニック動物
US5767337A (en) * 1995-07-31 1998-06-16 Duke University Creation of human apolipoprotein E isoform specific transgenic mice in apolipoprotein deficient "knockout" mice
JP3507884B2 (ja) * 2000-03-07 2004-03-15 新潟大学長 遺伝子発現を指標とする統合失調症の客観的診断法
EP1364578A4 (fr) * 2001-02-27 2004-09-15 Japan Government Animal modele atteint d'une maladie mentale de type schizophrenie, methode d'obtention dudit modele et utilisation
US9677869B2 (en) 2012-12-05 2017-06-13 Perimeter Medical Imaging, Inc. System and method for generating a wide-field OCT image of a portion of a sample
JP6456015B2 (ja) * 2013-07-19 2019-01-23 国立大学法人三重大学 閉塞性動脈硬化症モデル動物、るい痩研究用モデル動物、及び全身性アミロイドーシスモデル動物としての非ヒト哺乳動物
US10765094B2 (en) 2014-07-31 2020-09-08 Transgenic Inc. Inflammation reporter system
WO2019014767A1 (fr) 2017-07-18 2019-01-24 Perimeter Medical Imaging, Inc. Récipient d'échantillon pour stabiliser et aligner des échantillons de tissu biologique excisés pour analyse ex vivo
US20230148575A1 (en) * 2020-04-07 2023-05-18 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric il1b and/or il1a

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WO1988010118A1 (fr) * 1987-06-23 1988-12-29 Biogen N.V. Expression de proteines dans du lait
WO1991013979A1 (fr) * 1990-03-05 1991-09-19 President And Fellows Of Harvard College Souris transgenique surexprimant l'il-4 et procede d'utilisation

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Publication number Priority date Publication date Assignee Title
JPH02207727A (ja) * 1989-02-08 1990-08-17 Yuu Honshiyou トランスジェニックマウス

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988010118A1 (fr) * 1987-06-23 1988-12-29 Biogen N.V. Expression de proteines dans du lait
WO1991013979A1 (fr) * 1990-03-05 1991-09-19 President And Fellows Of Harvard College Souris transgenique surexprimant l'il-4 et procede d'utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch, Week 9039 Derwent Publications Ltd., London, GB; Class B04, AN 90-294644 XP002030369 & JP 02 207 727 A (YU H) , 17 August 1990 *
See also references of WO9503397A1 *

Also Published As

Publication number Publication date
WO1995003397A1 (fr) 1995-02-02
EP0724628A4 (fr) 1997-07-02
JPH09500533A (ja) 1997-01-21
CA2167581A1 (fr) 1995-02-02

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