WO1995014769A1 - Souris transgenique preparee a l'aide de chromosomes artificiels de levure et d'une recombinaison homologue - Google Patents

Souris transgenique preparee a l'aide de chromosomes artificiels de levure et d'une recombinaison homologue Download PDF

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WO1995014769A1
WO1995014769A1 PCT/US1994/013604 US9413604W WO9514769A1 WO 1995014769 A1 WO1995014769 A1 WO 1995014769A1 US 9413604 W US9413604 W US 9413604W WO 9514769 A1 WO9514769 A1 WO 9514769A1
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yac
mutation
mutant
human
gene
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PCT/US1994/013604
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English (en)
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John Hardy
Karen Duff
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University Of South Florida
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Priority to JP7515252A priority Critical patent/JPH09509046A/ja
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    • 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
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • 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)

Definitions

  • the present invention relates to transgenic nonhu an animals and specifically to methods of producing a mouse which expresses a mutant protein sequence.
  • YACs are large segments of DNA, typically of 100 kb to 1.5 Mb which are cloned into yeast. Because of the capacity of YAC vectors, they are used to clone large segments of DNA, including whole genes with intronic and distant regulatory elements. Unlike conventional transgenes, YACs direct expression with the correct temporal, spatial, and transcript distribution. This is accomplished because the genes are controlled by their native controlling elements, as opposed to inserted non-native elements used as promotors and regulators and the like with other transgenic techniques. These techniques have been applied to the APP gene 24 as well as the tyrosinase gene and the heavy chain im unoglobulin gene.
  • mice can be utilized as a model for Alzheimer's disease. Similar approaches could be used with other human diseases. Further, such mice can be used for testing potential drugs for various diseases having etiologies and pathologies related to the amyloid precursor protein.
  • a method of producing a mouse that expresses mutant protein sequences by obtaining a partial or whole gene containing a predetermined mutation which expresses mutant protein sequences and utilizing yeast artificial chromosome (YAC) transgenics to insert the gene into embryonic stem cells.
  • the mutation is introduced into the YAC by homologous recombination.
  • the stem cells are injected into blastocysts to derive a transgenic mouse which expresses the mutant protein sequences.
  • the present invention further provides a transgenic mouse made by the aforementioned method.
  • the present application further provides a transgenic mouse having a phenotype characterized by expressing mutant human amyloid precursor protein (APP) sequences.
  • APP human amyloid precursor protein
  • the present invention also provides mouse embryonic stem cells modified by the process of obtaining the partial or whole gene containing a predetermined mutation which expresses mutant protein sequences and utilizing YAC transgenics to insert the gene into embryonic stem cells thereby introducing the mutation into the mouse genome.
  • a mouse is derived which can be specifically utilized as an animal model of Alzheimer's disease by expressing mutant APP sequences which are otherwise found in humans suffering from Alzheimer's disease.
  • the present invention can be utilized either in the form of an animal model or even in the form of transformed cells in accordance with procedures well known to those skilled in the art as vehicles for testing potential drugs with regard to their pharmacology as well as utilizing well characterized drugs to test the physiological and biochemical parameters of the altered cells, all of these procedures being utilized to better characterize the pharmacodynamics of a human suffering from Alzheimer's disease.
  • Such tools are used by the pharmacologist as well as others, to design, derive, and test potential therapeutics.
  • Figure 1 is an experimental outline illustrating the outcome of the integrative (pop- in) and excisive (pop-out) recombination events conducted in accordance with the present invention
  • Figure 2 is a PCR analysis of pop-in clone C5 and pop-out clones C2 and C3, duplicate PCR reactions for each primer set were set up; one of each duplicate was then digested with BC1I;
  • Figure 2A shows lanes 1-7 pop-in clone C5: Lane 1 primer set 2 (Seq. ID NOs. 3,4) uncut. Lane 2, primer set 2 cut. Lane 3 primers 3R/2F (Seq. ID No. 6,5) uncut. Lane 4 primers 3R/2F, cut. Lane 5 primer set 3 (Seq. ID Nos. 5,6) uncut. Lane 6 primer set 3 cut. Lane 7 primer set 4 (Seq. ID Nos. 7,8) uncut;
  • Figure 2B shows lanes 8-15 pop-out clones 2 (mutant) and 3(wild type) : Lane 8 clone C2 primer set 2 uncut. Lane 9 clone C2 primer set 2, cut. Lane 10 clone C3 primer set 2 uncut. Lane 11 clone C3 primer set 2 cut. Lane 12 clone C2 primers 3R/2F uncut. Lane 13 clone C2 primers 3R/2F cut. Lane 14 clone C3 primers 3R/2F uncut, Lane 15 clone C3 primers 3R/2F cut.
  • Lane 16 1 kb ladder (BRL) ;
  • Figure 3 is a Southern blot analysis of YAC clones, the pop-out mutant clone C2 was run alongside the retrofitted YAC 9 and the native YAC (B142F9) undigested;
  • Figure 3A digested with BssHII ;
  • Figure 3B shows an electrophoresis alongside BioRad size markers (S_ ⁇ . cerevisiae and Lambda concatamers) showed that the undigested YACs are approximately 650 kb and that the BssHII fragment is approximately 200 kb.
  • the present invention generally provides a method of producing a mouse, or other non-human animal which expresses mutant protein sequences.
  • mammals are generally selected which closely mimic the human disease state and can be predictive of the pharmacodynamics and kinetics of potential therapeutics. Additionally, such animals can be utilized to study the development of disease as well as other aspects of disease and disease control.
  • the present invention preferably is directed to the production of a mouse model, although other animals could be used (eg. rats) .
  • the present invention is more specifically directed to the above-mentioned method for producing a mouse which expresses mutant human protein sequences.
  • Utility can be derived from the present invention in the generation of mouse models wherein the mutation is a human pathogenic mutation capable of expressing a human pathogenic protein sequence.
  • the invention is directed towards the characteristic pathology of Alzheimer's disease induced by APP mismetabolism.
  • Examples of specific mutations in the unrearranged human APP (hAPP) gene 20 are the pathogenic APP mutations APP670/1 17 and APP717Val- >Ile 18 .
  • other mutated genes can be used in accordance with the present invention to derive other mouse and other mammal models which express mutant protein sequences.
  • other sequences which can be used in accordance with the present invention which express mutant protein sequences are the mutant superoxide dismutase as a model for ALS and mutant CFTR as a model for cystic fibrosis.
  • Such partial or whole genes containing the predetermined mutation which expresses mutant protein sequences can thereby be obtained or generated.
  • the unrearranged hAPP gene is mutated using the "pop- in/pop-out" method as described below in detail and as set forth by Rothstein 16 , the techniques disclosed in the Rothstein reference being incorporated herein by reference.
  • the pop-in/pop-out method is an example of utilizing homologous recombination technology to insert a mutant gene fragment into a YAC.
  • YAC transgenics can then be utilised to introduce the mutant YAC into mouse embryonic stem cells and thence into mice.
  • URA3 has been used to select for integration events which lead to uracil prototrophy. Recombinational excision events are selected for by negative selection against URA3 gene using 5-fluoro-orotic acid (5-FOA) . This process requires that the host yeast strain be uracil dependent (requiring the enzyme orotidine- 5'-phosphate decarboxylase encoded by the URA3 locus) .
  • Efficient homologous recombination was found to occur within a 600 pb homologous region, linearised at a site 85 bp from the 3' end of the region and 118 bp from the mutation. Integration of the construct by a single recipocal crossover leads to duplication of the exon 17 region within the YAC.
  • the duplicated exon 17 regions can undergo a spontaneous intrachro osomal recombination event which, depending on the position of the crossover, leads to excision of either the mutant or the wild type exon.
  • other selection systems for assessing YAC recombination events can be used.
  • the vector used in the construction of most YAC libraries, pYAC4 also uses URA3 as the selectable marker. Therefore, the first stage in the protocol is the disruption of the URA3 locus in the vector arm which can be achieved with the retrofitting vector pRVl 18 . This procedure inserts a selectable marker for lysine prototrophy into the URA3 locus YAC arm. This thereby converts the yeast from uracil to lysine prototophy. The specific methodology is described below in the experimental section. Specific hAPP YACs have been made containing the APP670/1 mutation and containing the APP717 mutation.
  • YACs can be made incorporating other mutations as well as combinations of mutations, such as the combination of the APP670/1 and APP717 mutation.
  • An example of a YAC which can be used in accordance with the present invention is B142F9 (St. Louis YAC Library nomanclature) .
  • the YAC B142F9 was previously shown to contain the entire human APP gene, together with about 200 kb of upstream and 150 kb of downstream sequence 14 ' 16 .
  • B142F9 is used as an example, in principle any YAC can be so modified.
  • the ura3 gene in the vector arm is disrupted to convert YAC containing yeast from uracil to lysine prototrophy.
  • Exon 16 containing mutations at codons APP670 and 671 and exon 17 containing mutations at APP717 are cloned into a yeast shuttle vector containing the ura3 gene (eg. pRS406) , using sequence information derived from Yoshikai et al. 20 .
  • the YAC containing yeast are transformed with the linearized construct and selected in uracil free media (pop-in event) . Pop-out events are selected utilizing 5-FOA, as specifically described below.
  • YACs are selected which contain the mutation and are otherwise unrearranged.
  • the YAC-containing yeast is grown up in nutrient broth and prepared for pulsed-field electrophoresis as described 21 .
  • the YAC and other yeast chromosomes are separated by pulse field gel electrophoresis.
  • the YAC is cut out of the gel and the gel-agarose digested with beta- agarase. Care is taken at all stages so that the YAC is not sheared.
  • Cl cells can be used which are cultured. Examples of other stem cells which can be used are AB1, D3, E14 or any other ES cell capable of contributing to the germline in accordance with the present invention. Of course, other cell lines can be used by those skilled in the art.
  • the cells are maintained in culture on neomycin resistant fibroblast feeder cell layers 22 . The rationale for using neomycin resistant fibroblast lines is that these feeder cells will survive the selection procedures (see below) .
  • the stem cells are transferred to 35-mm wells the day before lipofection. 800ng of purified YAC DNA together with 12ng pPGK-neo
  • telomere (selectable marker) is allowed to complex with lO ⁇ g of TransfectamTM (Promega) diluted to lOO ⁇ l with OptiMEMTM (BRL) for one hour in plastic tubes.
  • the YAC/pPGK-neo/lipid complex is diluted 10 fold with ES media (BRL) and added to the drained ES cells. These are incubated overnight.
  • the rationale of this procedure is that a proportion of the cells will take up the DNA, including both the plasmid conferring neomycin resistance and the YAC DNA. Neomycin resistant cells can then be selected.
  • G418 a neomycin analog which is toxic in the absence of a neomycin-resistance gene
  • Resistant cells are picked and expanded. Nearly all of these cells will have incorporated therein the neomycin resistance gene (encoded by pPGK-neo) . A proportion of the cells will also have incorporated therein the YAC.
  • These cells are then screened for incorporation of YAC DNA by PCR screening for the presence of human exon 17. This is an efficient screening protocol which means that each colony does not have to be screened individually and thus allows many colonies to be screened rapidly. Positive colonies are then screened for other exons of APP by methods previously described to assess whether the whole YAC has been incorporated into the cells 23 .
  • the initial screen for the integrity of the YAC is done by PCR methods; first, through PCR of selected APP exons and the APP promoter 8 ' 23 . Also ALU-PCR (which gives PCR bands only in the presence of human DNA) is carried out and the ALU-PCR profile of the stem cells is compared to the profile of the original YACs.
  • Cell lines which appear to contain the whole hAPP gene are further characterized to see whether they contain the entire YAC (by screening the YAC vector arms) .
  • Cell lines which contain all the hAPP exons and promoter are subjected to pulsed field gel electrophoresis so that the gene can be mapped by hybridization of an APP probe to immobilized DNA and compared to the map of the gene in the YAC.
  • the cell lines are also examined for expression of the hAPP gene by SI nuclease protection assay for hAPP specific sequences including sequences that allow the differential assay of hAPP695, hAPP751 and hAPP770 from their mouse equivalents, as described below.
  • the relative ratios of the different human isoforms are compared with the ratios of the mouse isoform. This experiment gives some indication of whether the mouse cells are processing the hAPP transcript in the same way as they process the mouse transcript.
  • Mouse strain C57 blastocysts are isolated by flushing oviducts. The blastocysts are injected with stem cells and the resultant blastocysts transferred to pseudopregnant C57 foster mothers by standard techniques 22 . Coat color is used as a screen to determine which animals are likely to have a high proportion of cells derived from the stem cells since C57 mice are black but the line from which the stem cells are derived is agouti in color. The mice which appear to have a large amount of stem cell derived component will be mated with FVB mice and coat color used to determine which animals were derived from the stem cells. This breeding protocol has been chosen so that the C57 mice do not contribute to the germ line of the transgenic animals because Price and others have shown that aged C57 mice have inclusions in their brains which can be mistaken for amyloid deposits.
  • mice derived from the stem cells are analyzed in at least the following ways:
  • the integrity of the hAPP gene is analyzed by PCR and by pulse-field gel electrophoresis as described above for the stem cells.
  • the tissue-specific cell pattern of expression of hAPP is analyzed by SI nuclease protection 29 and by RT-PCR 29 .
  • the expression of the hAPP gene is compared with that of the native gene in the same samples to determine whether the hAPP gene is being processed in the same way as the native gene.
  • the mice are examined for pathology at three months, nine months and 18 months, for "human-like" neuropathology.
  • stem cells can be made for either incorporation into blastocysts and derivation of transgenic animals, such as the transgenic mouse discussed above utilizing the specific method discussed above, or mutant YAC containing cells (eg. Cos) can be utilized for pharmacodynamic, phar acokinetic and other pharmacology and related testing procedures by those procedures well known in the art.
  • mutant YAC containing cells eg. Cos
  • PCR was used to amplify a portion of genomic DNA from an affected family (F172) member carrying the valine-isoleucine mutation of codon 717 of APP transcript 770.
  • Primer set 1 (Seq. ID. Nos. 1,2) (Table 1) was used in a standard lOO ⁇ l PCR mix cycled as follows: 90°C five minutes, 94°C one minute, 54°C one minute, 72°C one minute, (x35 cycles), 72 ⁇ C ten minutes.
  • PCR products from several reactions were pooled and purified using MagicTM PCR preps (Promega) .
  • the amplimer was digested for three hours with Hindlll and the product purified on a MagicTM clean up column.
  • the pRS406 vector DNA (Stratagene) was also digested with Hindlll for three hours and the reaction terminated by incubation at 65°C for ten minutes. Subsequent recircularisation of the vector was prevented by CIAP treatment (Promega) at 37 ⁇ c for a total of 50 minutes. Approximately equal amounts (200 ng) of the vector and insert were ligated using a T4 DNA ligase Ready-to-GoTM kit (Pharmacia) for 45 minutes at 16°C.
  • DH5 ⁇ subcloning efficiency competent cells BBL
  • the whole transformation mix (with the volume reduced to lOO ⁇ l) was spread on L agar plates containing ampicillin (lOO ⁇ g/ml) , X gal and IPTG (Promega) LB.
  • White colonies were gridded for future reference and screened by direct PCR of a toothpicked colony using primer set 2 (Seq. ID Nos. 3,4).
  • the presence of the mutation was confir ed by digestion of the unpurified PCR product with Bell which cleaves the product into two equal sized fragments. The digestion products were visualized on a 2% MetaphorTM (FMC)/1% agarose gel.
  • FMC MetaphorTM
  • the mutant colonies were grown up in TB supplemented with ampicillin (50 ⁇ g/ml) and the plasmid DNA purified using MagicTM miniprep columns (Promega) .
  • the DNA was digested with 0.5 U of both Sail (Promega) and CellI (Boehringer) in Boehringer buffer H to assess the orientation of the fragment.
  • Genomic DNA is prepared by standard phenol/chloroform extraction procedures or by using column based methods 29 .
  • One of the mutant clones was sequenced in both directions along its entire length.
  • the double stranded template DNA was prepared 25 and sequenced using a Sequenase* 2.0 kit (USB). Products of the reaction were resolved by electrophoresis through a 6% SequagelTM polyaerylamide gel (National Diagnostics) . Sequencing primers (at 2 uM) are those listed in Table 1.
  • Transformants which had undergone integration of the APP construct were screened by standard PCR with primer sets 2 (Seq. ID Nos. 3,4), 3 (Seq. ID Nos. 5,6) and 4 (Seq. ID Nos. 7,8) (Table 1) using the conditions outlined previously.
  • YAC9 and mutant YAC C2 were prepared (vide supra) .
  • the yeast chromosomes were separated by PFGE (BioRad CHEF III®) through a 1% gel, with a switch time of 50-90 seconds at 6V/cm for 25 hours at 14°C.
  • the DNA was blotted onto HybondTM N+ and hybridized for one hour at 68 ⁇ C in Quick- hybTM (Stratagene) with purified APP PCR product amplified with primer set 2.
  • the probe had been labelled for 10 minutes at 37°C to high specific activity using a Ready-to-GoTM DNA labeling kit (Pharmacia) .
  • the blot was washed in 1 x SSC
  • primers 2F and 2R are located within the exon and therefore amplify a fragment of 385 bp using both the mutant and endogenous exon as a template. Digestion of the PCR product with the selective enzyme (Bell) 9 showed that both the mutant and wild type sequences were present. However, the mutation is almost equidistant from primer sites 2F and 2R and it was not possible to resolve the two fragments generated by the digestion.
  • the sequence of primer 3R (Seq. ID No. 6) is located outside of the mutant construct and therefore recognizes the APP sequence in the YAC. When combined with a primer specific for the vector (primer 3F, Seq. ID No.
  • the pop-out event occurs spontaneously and was selected for directly by plating pop-in clone C5 on agar lacking lysine but containing 5FOA. Only colonies which have undergone the pop-out (excision event) will have lost the vector encoded URA3 gene and can grow on 5F0A plates. Five such colonies were selected for further analysis.
  • the pop-out clones were initially screened by amplification with primer set 2 (Seq. ID Nos. 3,4) followed by digestion of the product with Bell. Two of the five clones failed to amplify with the APP primers, suggesting loss of the exon 17 region. The remaining three clones generated a PCR product with primer set 2.
  • the integrity of the mutant YAC was assessed at several levels.
  • agarose plugs prepared from the original B142F9 and the retrofitted clone 9 were electrophoresed alongside the mutant YAC Cl.
  • the DNA was blotted and hybridized with an APP exon 17 PCR probe.
  • the probe hybridized to a single band of equivalent size in all three samples assessed as approximately 650 kb (Fig. 3A) .
  • the three agarose plugs were digested with BssHII and electrophoresed as described above.
  • the exon 17 probe hybridized to a single band of approximately 200 kb in all three samples (Fig. 3B) .
  • Third, PCR direct sequencing of the mutant YAC was carried out to check that the mutation was the only change inserted into the YAC during this procedure.
  • the present invention illustrates that targeted homologous recombination in yeast is a viable and comparatively rapid alternative to gene targetting directly in embryonic stem cells as discussed above with regard to the prior art.
  • the procedure is fast (the experiments were conducted by a small team in three months) , cheap (only standard molecular biologically equipment are used) , and requires only that a minimal amount of sequence information (less than 1 kb) is known around each mutation site.
  • sequence information less than 1 kb
  • more than 5 kb of isogenic sequence information is required for efficient homologous recombination in mammalian cells using the hit and run protocol 4 . This usually requires the isolation and characterization of mouse homologs of human genes.
  • YAC utagenesis is also applicable to all human or murine genes capable of being cloned in yeast. It is expected that the process can also be used for other mammalian genes. Also of note is the fact that, through the human genome project, YACs from many genes are becoming centrally available. Thus, the ability to carry out these procedures is not limited by the availability of the molecular resources.
  • the present invention provides a useful method for producing an animal, such as a mouse, which expresses mutant protein sequences, such as mutant APP, using YAC transgenics.
  • a transgenic mouse made by the method is described above and having a phenotype characterized by expressing mutant human APP sequences.
  • the present invention described above provides mouse embryonic stem cells modified by the above described process.
  • Vecors for inserting selectable markers in vector arms and human DNA inserts of yeast artificial chromosomes (YACs). Gene, 103, 53-59.

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Abstract

Procédé de production d'une souris qui exprime des séquences de protéines mutantes humaines par: a) obtention d'un gène partiel ou entier contenu dans un chromosome artificiel de levure; b) introduction d'une séquence d'ADN mutant prédéterminé dans le chromosome artificiel de levure par recombinaison homologue; c) utilisation de méthodes transgéniques pour insérer le gène mutant dans des cellules souches embryonnaires, et d) injection des cellules souches dans des blastocystes afin d'en dériver une souris transgénique exprimant les séquences de protéines mutantes. Une souris transgénique obtenue par le procédé ci-dessus et des chromosomes artificiels de levures obtenus par le procédé ci-dessus sont également décrits.
PCT/US1994/013604 1993-11-29 1994-11-22 Souris transgenique preparee a l'aide de chromosomes artificiels de levure et d'une recombinaison homologue WO1995014769A1 (fr)

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WO1996040895A1 (fr) * 1995-06-07 1996-12-19 Athena Neurosciences, Inc. Procede d'identification de therapies de la maladie d'alzheimer a l'aide de modeles animaux transgeniques
WO1996040896A1 (fr) * 1995-06-07 1996-12-19 Athena Neurosciences, Inc. Procede pour identifier une therapeutique de la maladie d'alzheimer a l'aide de modeles animaux transgeniques
WO1997007227A2 (fr) * 1995-08-16 1997-02-27 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procede de modification ciblee de l'adn cellulaire
US5712134A (en) * 1990-05-09 1998-01-27 The Biological Research Center Of The Hungarian Academy Of Sciences Method of producing a cell carrying an excess of mammalian centromeres
US5811633A (en) * 1992-01-07 1998-09-22 Wadsworth; Samuel Transgenic mouse expressing APP770
WO1999009150A1 (fr) * 1996-08-15 1999-02-25 Bayer Corporation Methode permettant d'introduire des modifications dans un gene
EP0912722A1 (fr) * 1997-02-21 1999-05-06 Michael Nehls Procede servant a construire des vecteurs de recombinaison homologue pour une mutagenese dirigee
WO1999023239A1 (fr) * 1997-10-31 1999-05-14 Amgen Inc. Synthese efficace de vecteurs de ciblage genetique
US6025155A (en) * 1996-04-10 2000-02-15 Chromos Molecular Systems, Inc. Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
US6077697A (en) * 1996-04-10 2000-06-20 Chromos Molecular Systems, Inc. Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
US6335185B1 (en) 1998-02-03 2002-01-01 University Technologies International Inc. Bacteriophage vectors generated by bacteriophage/plasmid recombination
US6503712B1 (en) 2000-05-10 2003-01-07 Amgen Inc. Methods and compositions for preparing a genomic library for knockout targeting vectors
US6717031B2 (en) 1995-06-07 2004-04-06 Kate Dora Games Method for selecting a transgenic mouse model of alzheimer's disease
US6900026B2 (en) 1996-09-06 2005-05-31 Duke University Methods for identifying compounds as antioxidants
WO2007063160A2 (fr) * 2005-12-02 2007-06-07 Universidad Autónoma de Madrid Modele animal de la maladie d'alzheimer, procede d'obtention et applications correspondantes
WO2010051288A1 (fr) 2008-10-27 2010-05-06 Revivicor, Inc. Ongulés immunodéprimés
US7888104B2 (en) 2000-11-28 2011-02-15 Henkel Ag & Co. Kgaa Cyclodextrin glucanotransferase (CGTase), obtained from<I>Bacillus agaradherens<λ>(DSM 9948) and detergents and cleaning agents containing said novel cyclodextrin glucanotransferase
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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
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WO1996040895A1 (fr) * 1995-06-07 1996-12-19 Athena Neurosciences, Inc. Procede d'identification de therapies de la maladie d'alzheimer a l'aide de modeles animaux transgeniques
WO1997007227A2 (fr) * 1995-08-16 1997-02-27 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procede de modification ciblee de l'adn cellulaire
WO1997007227A3 (fr) * 1995-08-16 1997-04-10 Deutsches Krebsforsch Procede de modification ciblee de l'adn cellulaire
EP2314708A1 (fr) 1996-04-10 2011-04-27 Chromos Molecular Systems, Inc. Chromosomes artificiels, leurs utilisations et leurs procédés de préparation
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EP0912722A4 (fr) * 1997-02-21 2001-12-05 Michael Nehls Procede servant a construire des vecteurs de recombinaison homologue pour une mutagenese dirigee
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US7888104B2 (en) 2000-11-28 2011-02-15 Henkel Ag & Co. Kgaa Cyclodextrin glucanotransferase (CGTase), obtained from<I>Bacillus agaradherens<λ>(DSM 9948) and detergents and cleaning agents containing said novel cyclodextrin glucanotransferase
EP2527456A1 (fr) 2004-10-22 2012-11-28 Revivicor Inc. Porcs transgéniques déficients en chaîne légère d'immunoglobuline endogène
WO2007063160A2 (fr) * 2005-12-02 2007-06-07 Universidad Autónoma de Madrid Modele animal de la maladie d'alzheimer, procede d'obtention et applications correspondantes
WO2007063160A3 (fr) * 2005-12-02 2007-07-26 Univ Madrid Autonoma Modele animal de la maladie d'alzheimer, procede d'obtention et applications correspondantes
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