EP0799305A1 - Animal transgenique presentant une carence en proteine precurseur d'amyloide native - Google Patents

Animal transgenique presentant une carence en proteine precurseur d'amyloide native

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Publication number
EP0799305A1
EP0799305A1 EP95942534A EP95942534A EP0799305A1 EP 0799305 A1 EP0799305 A1 EP 0799305A1 EP 95942534 A EP95942534 A EP 95942534A EP 95942534 A EP95942534 A EP 95942534A EP 0799305 A1 EP0799305 A1 EP 0799305A1
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Prior art keywords
app
mouse
gene
altered
mice
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German (de)
English (en)
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EP0799305A4 (fr
Inventor
Hui Zheng
Howard Y. Chen
Myrna E. Trumbauer
Leonardus H.T. Van Der Ploeg
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Merck and Co Inc
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Merck and Co Inc
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Publication of EP0799305A4 publication Critical patent/EP0799305A4/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Withdrawn legal-status Critical Current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
    • A01K67/0276—Knock-out 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
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/02—Cells from transgenic animals

Definitions

  • the present invention relates to a transgenic nonhuman animal lacking native amyloid precursor protein.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • senile plaques consist of extracellular deposits containing a ⁇ -amyloid core surrounded by a halo of dystrophic neurites, glia and astrocytes. ⁇ -amyloid deposits are present in neocortex blood vessel walls.
  • the major component of senile plaques is a 4 kDa peptide referred to as A ⁇ , that is proteolytically cleaved from a larger 120 kDa amyloid precursor protein (APP).
  • a ⁇ amyloid precursor protein
  • Other components of the plaques include ubiquitin, amyloid P, Apo E, interleukin- 1 , and ⁇ -1 -antichymotrypsin.
  • APP amyloid precursor protein
  • FAD early onset familial AD
  • Genetic analysis of FAD families has established that the disorder is inherited as a dominant autosomal gene defect, which maps to the long arm of chromosome 21 and is closely linked to the APP gene. These findings are consistent with genetic data obtained from the analysis of Down syndrome patients.
  • FAD families have also been identified in which an early onset of AD is strictly correlated with the presence of a mutation in exon 17 of the APP gene at amino acid 717 (Val-Ile). This mutation within the transmembrane spanning domain of the APP co- segregates with FAD.
  • the APP gene is approximately 400 kb in length and encodes a glycosylated, transmembrane protein which may be involved in cell-cell interaction.
  • the APP gene has at least 18 exons that create at least 5 distinct APP transcripts by alternative splicing.
  • the predominant transcripts encode proteins of 695, 751 and 770 amino acids (these major forms of APP are referred as APP695, APP751 and APP770, respectively).
  • Transcripts for APP695 are enriched in the brain.
  • Transcripts encoding APP751 and APP770 mRNA species predominate in peripheral tissues. All three isoforms contain the 42 amino acid A ⁇ domain.
  • APP isoforms 751 and 770 contain an additional 56 amino acid insert encoding the Kunitz type serine protease inhibitor (KPI).
  • KPI Kunitz type serine protease inhibitor
  • APP is proteolytically metabolized by at least two pathways. One pathway involves an ⁇ -secretase cleavage site positioned between Lys 16 and Leu 17 of A ⁇ domain; proteolytic cleavage at this site precludes the formation of an amyloidogenic A ⁇ entity. The second pathway produces intact, amyloidogenic A ⁇ (39-42 amino acids) by proteolytic cleavages at the ⁇ - and ⁇ -secretase cleavage sites of the full- length APP molecule.
  • AD patients are also found in aged humans and other aged mammals including non-human primates, polar bears and dogs.
  • other aged mammals such as laboratory rats and mice, do not normally develop A ⁇ deposits. This could be due to the fact that the three amino acid differences present in the ⁇ -amyloid sequence between human and mouse APP prevents mouse A ⁇ from forming plaques.
  • the lack of a cost-effective, experimental animal model mimicking human pathogenesis hinders the understanding AD neuropathology and developing therapeutics against AD.
  • Transgenic technology may offer a suitable alternative to this problem.
  • Addition of a gene construct directing high levels of human APP or its components to key regions in the murine central nervous system may cause neuropathological changes resembling the AD phenotype.
  • Attempts to express human amyloid precursor protein segments or the full-length wild type protein in transgenic animals have been successful.
  • transgenic mice of the present invention are useful in the determination of the in vivo function of APP and the ⁇ - amyloid peptide in the central nervous system and in other tissues. These mice are being bred with transgenic mice expressing the human APP FAD with the aim of producing a strain of mice in which the only APP produced is of human origin. The precise roles of APP in AD is not fully understood at this time. Due to the biological importance of APP in AD and other neurological disorders, the APP gene is an important target for embryonic stem (ES) cell manipulation.
  • ES embryonic stem
  • APP deficient transgenic mice would aid in defining the normal role(s) of APP, and allow an animal model of APP deficiency to be used in the design and assessment of various approaches to modulating APP activity.
  • Such APP modified transgenic mice can also be used as a source of cells for cell culture.
  • the present invention relates to a transgenic nonhuman animal lacking native amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • the transgenic mouse of the invention may be used in the study of Alzheimer's Disease and disorders involving the central nervous system. BRIEF DESCRIP ⁇ ON OF THE DRAWINGS
  • Figure 1 is a genomic map of the mouse APP gene, the location of the cosmid clone isolated from a primary genomic library and different probes and subclones generated from the cosmid clone.
  • ex 1 exon 1 of the APP gene.
  • Figure 2 is the predicted modification of the mouse chromosomal APP gene by targeted recombination using replacement vector pHZ 038.
  • the targeting vector (pHZ038) contains from left to right:
  • the probes used for Southern blot analysis were the 1.0 kb Xbal-Bglll fragment (5'-probe), the 0.8 kb Bglll-Ncol fragment (3'- probe), both of which are outside the targeting vector, and the neo sequence.
  • EcoRI was used to differentiate the wild-type and the targeted APP alleles, which generates a 9.0 kb and 6.5 kb fragments by the 5'-probe and a 9.5 kb and a 9.0 kb by the 3'-probe, respectively.
  • R EcoRI
  • X Xbal
  • B Bglll
  • N Ncol
  • Pr mouse APP promoter
  • El exon 1 of the mouse APP gene.
  • PGK phosphoglycerate kinase promoter.
  • Figure 3 is a Southern hybridization analysis of four targeted embryonic stem (ES) clones having an APP knockout.
  • ES cell DNA 8 ⁇ g from the wild-type AB2.1 cells and four positive clones (76, 123, 174 and 196) were restriction enzyme digested with EcoRI, electrophoresed on a 0.7% agarose gel, transferred onto a Gene Screen Plus nylon membrane (NEN-Dupont) and hybridized with a 5'-probe (labeled a), a 3'-probe (data not shown), and a neo probe (labeled b). A 6.5 kb diagnostic fragment is detected by the 5'-probe in all the targeted clones in addition to the wild-type 9.0 kb fragment.
  • Probing of the same filter with a neo coding sequence shows that the neo gene is present in the 6.5 kb band and is the only integration event in all the clones.
  • the other two bands at high molecular weight corresponds to a nonfunctional neo sequence introduced by a retrovirus in the parental AB2.1 cells.
  • Figure 4 is a Southern hybridization analysis of tail DNA from transgenic mice having an APP knockout. Southern analysis of genomic DNA from het. x het. crosses yielded the expected number of mice homozygous for the disrupted APP allele.
  • Genomic DNA isolated from the tails of two week old pups generated from crosses of heterozygous mice was digested with EcoRI, blotted onto filters, and hybridized with the 5'-probe. +/+: wild-type; +/-: heterozygotes; -/-: homozygous APP deficient mice.
  • Figure 5 is a Northern hybridization analysis for the determination of APP transcripts in the knockout and wild-type control mice. As expected, the brain RNA from the knockout mice did not exhibit any detectable APP expression, whereas wild-type control and heterozygous animals showed a significant amount of APP activity.
  • RNA 20 ⁇ g from wild-type (+/+), heterozygous(+/-) and homozygous (-/-) APP mice were isolated (2 mice each) from brain using the RNAzol B method (Biotecx
  • the present invention relates to a transgenic nonhuman animal lacking native amyloid precursor protein.
  • the transgenic mouse of the invention may be used in the study of Alzheimer's Disease and disorders involving the central nervous system.
  • a 39 to 43 amino acid ⁇ -amyloid peptide is the major component of the neuritic plaques characterizing Alzheimer disease, ⁇ - amyloid is derived from a larger amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • the APP mRNA undergoes alternative splicing to generate several isoforms, encoding proteins that range from 695 to 770 amino acid residues. Among these, APP695 is expressed predominantly in neurons and APP751 and APP770 can be detected in all the tissues examined.
  • Five different types of point mutations have now been identified in the human APP gene, causative of familial early onset Alzheimer's disease (FAD) in several unrelated families. These affected families provide the strongest evidence yet for the notion that APP processing and the ⁇ - amyloid peptide serve a central role in Alzheimer disease progression.
  • APP is one of the most abundant proteins in the brain and the ⁇ -amyloid peptide is secreted in cerebrospinal fluid (CSF) of healthy individuals and AD patients.
  • CSF cerebrospinal fluid
  • the functions of the APP and ⁇ -amyloid in vivo are obscure.
  • the APP has been implicated as a growth factor in vitro in fibroblast cultures.
  • the ⁇ -amyloid peptide has been shown to have neuroprotective and neurotoxic actions, dependent on the cell line and protein preparations tested.
  • a Kunitz protease inhibitor domain in the N-terminal portion of the APP may serve a role in regulating protein half-life.
  • mice may be useful as acceptors of the human FAD protein.
  • Mouse APP is overall conserved when compared to human APP but differs in three potentially essential amino acid residues of the ⁇ -amyloid domain.
  • the murine APP gene is about 400 kb in size and is encoded by at least 18 exons.
  • the murine APP gene was inactivated by deleting its promoter and first exon, which encodes the ATG translation initiation codon. To target the APP gene in murine ES cells, a positive-negative selection strategy was used.
  • the targeting vector pHZ038 ( Figure 2) encoded 8.5 kilobases (kb) of DNA derived from the 5' end of the APP gene.
  • a 3.8 kb sequence encoding the APP promoter and the first intron was deleted from this vector and replaced with a positive selectable marker, PGKneo (neomycin-phospho-transferase).
  • a MC1 - TK (thymidine kinase) cassette (labeled HSV-TK in Figure 2) was inserted at the end of the vector for negative selection. Correct homologous recombination between the targeting vector and one of the APP alleles in the ES cells would result in a deletion of the APP promoter and exon 1 , encoding the signal peptide.
  • the targeting vector was electroporated into AB2.1 ES cells. G418 and FIAU resistant clones were screened by a mini- Southern protocol. A five-fold enrichment was achieved by selecting the cells with FIAU. Six targeted clones were identified and the frequency of targeted recombination versus random integration at the APP locus was 1/160 ( Figure 3). Of four clones injected into blastoysts two (no. 76 and 174) transmitted the targeted APP allele to the offspring. Heterozygous matings were set up to produce mice homozygous for the disrupted APP gene.
  • mice Homozygous APP knockout mice that resulted from these breedings were generated at expected frequencies (Figure 4). These mice appeared normal and healthy up to 14 weeks of age. Northern blot analysis of RNA isolated from brain using APP695 cDNA as a probe showed that APP mRNA was not produced in mice homozygous for the targeted allele. The APP mRNA level was reduced by approximately 50% in the heterozygous mice as compared to wild-type controls ( Figure 5).
  • the present invention utilizes a cloned DNA encoding the
  • Transgenic animals are generated which have an altered APP gene.
  • the alterations to the naturally occurring gene are modifications, deletions and substitutions. Modifications, deletions and substituteions may render the naturally occurring gene nonfunctional, producing a "knockout" animal, or may lead to an APP with altered function.
  • These transgenic animals are critical for drug antagonist or agonist studies, for creation of animal models of human diseases, and for eventual treatment of disorders or diseases associated with APP.
  • Transgenic animals lacking native APP are useful in characterizing the in vivo function of APP.
  • a transgenic animal carrying a "knockout" of APP is useful for the establishment of a nonhuman model for diseases involving APP, and to distinguish between the activities of APP in in vivo and in vitro systems.
  • 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 an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection 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 genetic alteration or genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
  • the altered APP gene should not fully encode the same APP as native to the host animal, and its expression product should be altered to a minor or great degree, or absent altogether. However, it is conceivable that a more modestly modified APP gene fall within the scope of the present invention.
  • ES cells may be obtained from pre- implantation embryos cultured in vitro and fused with embryos (M. J. Evans ai-, Nature 292: 154-156 (1981); Bradley et ah, Nature 309: 255-258 (1984); Gossler et aL Proc. Natl. Acad. Sci. USA 83: 9065-9069 (1986); and Robertson et aj., Nature 322, 445-448 (1986)).
  • Transgenes can be efficiently introduced into the ES cells by a variety of standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction.
  • the resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
  • the introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (R. Jaenisch, Science 240: 1468-1474 (1988)).
  • APP functions are complex, they must be examined in a variety of ways.
  • One approach to the problem of determining the contributions of individual genes and their expression products is to use isolated genes to selectively inactivate the native wild-type gene in totipotent ES cells (such as those described herein) and then generate transgenic mice.
  • the use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described 1987 (Thomas et a] . ., Cell 51 :503-512, (1987)) and is reviewed elsewhere (Frohman et al., Cell 56:145-147 (1989); Capecchi, Trends in Genet. 5:70-76 (1989); Baribault et aL, Mol. Biol. Med.
  • Nonhomologous plasmid-chromosome interactions are more frequent, occurring at levels 105-fold (Lin et ah, Proc. Natl. Acad. Sci. USA 82:1391-1395 (1985)) to 102-fold (Thomas et al-, Cell 44:419-428 (1986); Song et aL, Proc. Natl. Acad. Sci. USA 84:6820-6824 ( 1987)) greater than comparable homologous insertion.
  • PCR polymerase chain reaction
  • a positive genetic selection approach has been developed in which a marker gene is constructed which will only be active if homologous insertion occurs, allowing these recombinants to be selected directly (Sedivy et al-, Proc. Natl. Acad.
  • PNS positive-negative selection
  • Nonhomologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with the herpes drugs such as gancyclovir (GANC) or FIAU (l-(2-deoxy 2-fluoro-B-D-arabinofluranosyl)-5-iodouracil).
  • HSV-TK Herpes Simplex virus thymidine kinase
  • GANC gancyclovir
  • FIAU l-(2-deoxy 2-fluoro-B-D-arabinofluranosyl
  • a “targeted gene” or “Knock-out” is a DNA sequence introduced into the germline of a non-human animal by way of human intervention, including but not limited to, the above described methods.
  • the targeted genes of the invention include DNA sequences which are designed to specifically alter cognate endogenouos alleles.
  • the methods for evaluating the targeted recombination events as well as the resulting knockout mice are readily available and known in the art. Such methods include, but are not limited to DNA (Southern) hybridization to detect the targeted allele, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots to detect DNA, RNA and protein.
  • genomic libraries were constructed from ES cells grown in the absence of feeder cell layers for the isolation of genes to be used for subsequent ES cell targeting. Genomic libraries were prepared from AB2.1 cells according to the in situ procedure described in (Mudgett et ah, Genomics 8:623-633, (1990)).
  • the cosmid vector sCos-1 was chosen, as it allows both the vector and the insert to be dephosphorylated. This prevents concantamer formation and generally results in genomic libraries of better quality and quantity (up to 5x106 clones per package) than is achieved with other vectors (Evans et ah, Gene 79:9-20, (1989)).
  • the genomic library was constructed with bacterial hosts SURE.
  • the SURE host line (Stratagene) was used to stably maintain indirect repeats and allow isolation of methylated DNA.
  • the primary mouse cosmid library of Example 1 was screened using a 1.0 kb Hindlll-PvuTJ fragment located in the promoter of the APP gene as a probe (Izumi ______ Gene 112: 189-195 (1992)).
  • the cosmid DNA was digested with different restriction endonucleases, electrophoresed on agarose gels, and hybridized with different probes isolated from the plasmid HH5.0 (Izumi et al.. Gene 112: 189-195 (1992)) to map the boundary as well as the promoter and exon 1 of the mouse APP gene (Fig.l).
  • a gene targeting vector for inactivating the APP gene was prepared using standard cloning techniques (Sambrook et al.. supra): a). A three-way ligation was performed using the 1.4 kb Bglll-Xhol fragment in the 5' portion of the APP cosmid clone upstream of exon 1 (ex 1), the 7 kb Xhol-Bglll fragment in the 3' portion of the cosmid clone, and BamHI digested pKS vector. The resulting plasmid was named pHZ036. b).
  • Plasmid pHZ036 was partially-digested with Xhol and ligated with the 1.5 kb Xhol-Sall fragment of PGKneo. A ligation product with neo inserted in between the two APP fragments was selected and referred to as pHZ037. c). A 2 kb Xhol fragment from pKS-TK was inserted into the Sail digested pHZ037 vector. The resulting plasmid, pHZ038, is the complete construct for targeting of the mouse APP gene.
  • the targeting vector used in the APP gene disruption experiments was the pHZ038 vector of Example 3.
  • APP KO wild-type APP allele to generate the APP knockout
  • the mouse embryonic stem cell line AB2.1 was electroporated with NotI digested pHZ038 to linearize the plasmid, leaving the pks sequence attached to the end of the TK cassette. All AB2.1 ES cells were cultured on SNL feeder cells as described (Robertson, in Teratocarcinomas and embryonic stem cells, E L Press, pp. 71-112 (1987)).
  • Electroporations were performed with 1x107 ES cells and 25 ⁇ g linearized vector in 0.8 ml PBS buffer at 230v, 500 ⁇ F using a Bio-Rad Gene Pulser.
  • ES cell transformants were selected with the antibiotic geneticin (Gibco G418: 200 ⁇ g/ml active G418) 24 hr post electroporation, and some transformants were counter-selected with FIAU (Bristol Myers Squibb; 0.4 ⁇ M) 48 hours later for enhancement of homologous recombinants.
  • FIAU Bacillus Squibb
  • Murine leukemia inhibitory factor (LIF; ESGRO, Gibco BRL, Inc.) was used at 200 U/ml.
  • G418- and FIAU -resistant ES clones were isolated, grown up and analyzed by a mini-Southern protocol (Ramirez-Solis, R. et al. Anal. Biochem. 201:331-335, 1992). A total of six targeted clones were identified from 200 double resistant colonies analyzed. Therefore, the frequency of targeted recombination vs. random integration at the APP locus is 1/160.
  • ES cell line AB2.1 is homozygous for the agouti (A) coat color gene
  • penetrance of ES cells into the injected (black coat color) C57B1/6 blastocyst gives rise to chimeric coat color mice.
  • the chimeric coat color mice were bred to wild-type C57BL/6 (black coated) and 129/J (agouti coated) female mice. Some of the progeny from the chimera X C57BL/6 cross were expected to be agouti if the chimeric male had ES cell genetic material incorporated into its germline (agouti is dominant to black coat color). The chimera X 129/J cross would yield only agouti mice. These crosses were performed to transfer ES cell genetic information, including the disrupted APP allele, to its offspring. Breeding of three male chimeras from both clone 76 and 174 resulted in agouti pups when crossed with C57B1/6J females.
  • genomic DNA was purified from about 1 cm of tail taken from each mouse at about two weeks of age. The genomic DNA was isolated as described (Laird et al., supra), followed by phenol hloroform extractions and ethanol precipitation. Southern hybridization analysis (as described in Example 5) were used to identify offspring which contained the disrupted APP allele. These transgenic offspring were heterozygous for the APP disruption. Both transgenic heterozygous and nontransgenic mouse (tail) genomic DNAs were digested with EcoRI, and were hybridized with 5' flanking DNA probe to confirm the transgenic APP structure. Southern hybridization analysis confirmed that the structure of the altered APP allele was identical to that predicted, and previously characterized in the APP targeted ES clones. EXAMPLE 7
  • mice Male and female transgenic mice, each of which contained one copy of the altered APP allele (heterozygous mice), were mated with each other to generate mice in which both copies of the APP gene encoded the targeted, altered APP allele. It was predicted that one fourth of the mouse embryos would be homozygous for the altered APP gene.
  • Surviving offspring were genotyped by Southern hybridization as described above (Fig. 4). It was determined that 21 ( 24%) of the 87 offspring mice were homozygous APP-/-, 30 ( 34%) were wild-type APP+/+, and 36 (41 %) were heterozygous APP+/-. These numbers indicate that there was no significant decrease in the number of APP deficient transgenic mice which survived at two weeks of age.
  • mice of Example 7 Surviving homozygous APP deficient mice of Example 7 were bred with wild-type or heterozygous mates to determine if they were fertile. All homozygous APP-/- males and females tested were fertile. Significant differences in gross morphology or histology between the APP deficient mice and the wild-type or heterozygous mice were not observed.
  • the transgenic animals of the invention may be used as a source of cells for cell culture.
  • Cells of brain tissues lacking the APP gene may be cultured using standard culture techniques.

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Abstract

L'invention concerne un animal transgénique non humain, présentant une carence en protéine précurseur d'amyloïde native. La souris transgénique selon l'invention peut être utilisée dans l'étude de la maladie d'Alzheimer et de troubles impliquant le système nerveux central.
EP95942534A 1994-12-05 1995-12-01 Animal transgenique presentant une carence en proteine precurseur d'amyloide native Withdrawn EP0799305A4 (fr)

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US6717031B2 (en) 1995-06-07 2004-04-06 Kate Dora Games Method for selecting a transgenic mouse model of alzheimer's disease
WO1999060100A1 (fr) * 1998-05-15 1999-11-25 Ortho-Mcneil Pharmaceutical, Inc. Animaux transgeniques porteurs d'un gene irak modifie

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WO1993014200A1 (fr) * 1992-01-07 1993-07-22 Tsi Corporation Modeles d'animaux transgeniques utilises pour tester des traitements potentiels relatifs a la maladie d'alzheimer

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WO1993014200A1 (fr) * 1992-01-07 1993-07-22 Tsi Corporation Modeles d'animaux transgeniques utilises pour tester des traitements potentiels relatifs a la maladie d'alzheimer

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G. Wright et al., 1991, "High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep", Biotechnology, vol. 9, pages 830-834. *
LANNFELT L. ET AL.: "Alzheimer's disease: molecular genetics and transgenic animal models" BEHAVIOURAL BRAIN RESEARCH, vol. 57, no. 2, 1993, pages 207-213, XP002121614 *
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See also references of WO9617926A1 *
T. Yamada et al., 1987, "Complementary DNA for the mouse homolog of the human amyloid beta protein precursor", Biochemical and Biophysical Research Communication, vol. 149, pages 665-671. *

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