[go: up one dir, main page]

EP1182924A2 - SOURIS TRANSGENIQUE A GENE MARQUEUR LacZ REGULE PAR LE PROMOTEUR N-CAM SERVANT A L'IDENTIFICATION DE MODULATEURS N-CAM, METHODES ET COMPOSITIONS - Google Patents

SOURIS TRANSGENIQUE A GENE MARQUEUR LacZ REGULE PAR LE PROMOTEUR N-CAM SERVANT A L'IDENTIFICATION DE MODULATEURS N-CAM, METHODES ET COMPOSITIONS

Info

Publication number
EP1182924A2
EP1182924A2 EP99908469A EP99908469A EP1182924A2 EP 1182924 A2 EP1182924 A2 EP 1182924A2 EP 99908469 A EP99908469 A EP 99908469A EP 99908469 A EP99908469 A EP 99908469A EP 1182924 A2 EP1182924 A2 EP 1182924A2
Authority
EP
European Patent Office
Prior art keywords
cam
gene
cell
mouse
expression
Prior art date
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.)
Withdrawn
Application number
EP99908469A
Other languages
German (de)
English (en)
Other versions
EP1182924A4 (fr
Inventor
Kathryn L. Crossin
Gerald M. Edelman
Brent D. Holst
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scripps Research Institute
Original Assignee
Scripps Research Institute
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Scripps Research Institute filed Critical Scripps Research Institute
Publication of EP1182924A2 publication Critical patent/EP1182924A2/fr
Publication of EP1182924A4 publication Critical patent/EP1182924A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • 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
    • A01K2267/0393—Animal model comprising a reporter system for screening tests

Definitions

  • the invention relates a transgenic mouse having a N-CAM reporter gene construct useful for screening for N-CAM modulators, and to methods and compositions related to the transgenic mouse .
  • CAMs Cell adhesion molecules mediate neuronal and glial adhesion during development of the nervous system and thereby affect processes such as neurite fasciculation, axonal pathfinding, and synaptogenesis .
  • Edelman et al Annu . Rev . Biochem.. 60:155-190 (1991); and Edelman, G. M. , Dev. Dynam.. 193:2-10 (1992). It has been suggested that in the adult brain, regulation of adhesion alters the morphological and physiological properties of synapses through effects on membrane interactions. Lynch et al, (1991) In (Ascher, P., Choi, D. . & Christen, Y. , eds . ) .
  • N-CAM neural cell adhesion molecule
  • N-CAM The expression of N-CAM is therefor important from both the perspective of regulation of the gene encoding N-CAM and for the nature of the signaling pathways which affect N-CAM expression.
  • changes in various neuronal tissue expression of N-CAMs affects neurite fasciculation, axonal pathfinding and synaptogenesis, it is useful to have methods for the study of tissue distribution of expression of N-CAM.
  • the invention provides a system for characterizing N-CAM expression in vivo, for identifying tissue distribution of N-CAM expression under various physiologic, psychomotor and/or chemical stimuli, and for screening for bioactive molecules which modulate N-CAM expression.
  • the invention describes a transgenic mouse having a reporter gene under the control of the N-CAM promoter such that regulation of the N-CAM gene is reflected by regulated expression of the reporter gene in vivo .
  • which reporter gene is convenient to assay.
  • the N-CAM gene in the transgenic mouse has been modified by the insertion of the reporter gene between the transcription and translation initiation sites of the N-CAM gene of the mouse. The insertion places the expression of the reporter gene under the control of the N-CAM promoter.
  • the reporter gene is the bacterial lacZ gene.
  • the inserting is made in a single allele of the N-CAM gene, and through genetic crossing, one can produce a progeny mouse with the insertion in either a single N-CAM allele, i.e., heterozygous, or in both N-CAM alleles, i.e., homozygous .
  • the invention also describes methods for use of the transgenic mouse.
  • the transgenic mouse can be used to study tissue distribution of N-CAM expression by various analytical methods including in situ histological analysis of the expression pattern of the reporter gene, and in vitro detection of the reporter gene in various tissues, tissues sections and/or cell types.
  • the invention further contemplates a cultured cell derived from a transgenic mouse of the invention.
  • the cultured cell can be obtained directly from the mouse, from a descendant mouse, or can be a progeny of a primary culture of one or more cells of a mouse of this invention.
  • the cultured cell can be in the form of a single cell or cell line, or a composition of mixed cells.
  • the cultured cell can be obtained from a mouse which is the descendant of a transgenic mouse of this invention, such as by a cross with another mouse having either the same or a different genetic background.
  • a cell in the culture can be either homozygous or heterozygous for a reporter gene in the N-CAM allele.
  • a cultured cell of this invention has a variety of uses.
  • the cell can be used in vitro to study substances which affect N-CAM expression at the level of transcription or translation of the gene product.
  • Other uses will be apparent to one skilled in the art. Methods for culturing a cell from a mouse of this invention, and for using such a cultured cell, are also described.
  • Figures 1A-1E illustrate the overall strategy and details of targeted replacement of the N-CAM allele with lacZ, together with analysis of N-CAM expression in wild type, heterozygous and homozygous N-CAM knockout mice.
  • Figure 1A is a schematic diagram of the N-CAM locus in heterozygous animals after homologous recombination.
  • N-CAM promoter activity can be assayed both by ⁇ -gal activity and by quantitating N-CAM mRNA.
  • Figure IB illustrates the structures of the N-CAM targeting vector, wild-type N-CAM allele, and disrupted N-CAM allele. Restriction enzyme sites are abbreviated as: E, EcoRI ; K, Kpnl; N, NotI; S, Sail; Sa, SacII; X, Xhol .
  • E EcoRI
  • K Kpnl
  • N NotI
  • S Sail
  • Sa SacII
  • X Xhol
  • the hatched box above the diagram of the disrupted N-CAM allele indicates probe a (a Kpnl-Xhol intron 1 fragment) used for Southern blot analysis of the ES cell lines.
  • Figure IC illustrates a Northern blot.
  • Figure ID illustrates an ethidium bromide stain of the Northern gel of Figure IC prior to transfer and shows approximately equal loading of the total RNA.
  • Figure IE illustrates a Western blot of 30 ⁇ g total protein isolated from the brains of wild type (+/+), heterozygous (+/-) and homozygous (-/-) N-CAM knockout mice with a polyclonal antibody to N-CAM.
  • Figures 2A-2H illustrate ⁇ -gal expression in heterozygous and homozygous knockout mice in whole mount embryos and in tissue sections compared with N-CAM mRNA localization.
  • ⁇ -gal was expressed in the post-mitotic neurons of the hindbrain and the midbrain as well as the in the floor plate (Figure 2G) .
  • Figures 2F & 2H illustrate in si tu hybridization of E13.5 sections with N-CAM RNA probes. The expression of ⁇ -gal was similar to the pattern of N-CAM mRNA expression visualized by in si tu hybridization.
  • sc spinal cord
  • drg dorsal root ganglia
  • fp floor plate
  • mb midbrain
  • hb hindbrain.
  • Figures 3A-3G illustrate hippocampal morphology and physiology.
  • Figures 3A & 3B illustrate morphology of adult hippocampus using a hematoxylin and eosin stain of wild type ( Figure 3A, +/+) and homozygous ( Figure 3B, -/-) adult brain.
  • Figure 3C illustrates delivery of theta-burst stimulation (TBS) to path A (orthodromic) resulted in a rapid enhancement of fEPSP amplitudes that decayed over a brief period to a stable plateau. Approximately 1 hr later, application of the same high frequency stimulus to pathway B (antidromic) resulted in a comparable enhancement, without affecting established LTP in path A.
  • TBS ta-burst stimulation
  • pathway B antidromic
  • the initial period of enhancement after TBS is thought to reflect a mixture of post-tetanic potentiation and short-term potentiation, a more decremental form of NMDA receptor-dependent plasticity.
  • the brief heterosynaptic depression observed after TBS is likely related to adenosine release accompanying high-frequency stimulation, an effect commonly seen in hippocampal slices from normal mice.
  • Figure 3D illustrates an example of fEPSPs elicited with paired stimulation; interpulse interval equals 25msec.
  • Figure 3F illustrates a typical record of LTP induced in slices from knockout mice. Plotted in the upper and lower panels are amplitudes of fEPSPs elicited alternately at 0.1Hz along two independent pathways in the stratum radiatum of field CA1.
  • Figure 3G illustrates a plot of the average amount of LTP obtained in groups of slices from N-CAM knockout and wild type mice.
  • Figures 4A-4B illustrates ampakine induction of ⁇ -gal expression resulting from N-CAM promoter activity in heterozygous N-CAM knockout mice. ⁇ -gal expression was examined in sagittal sections from vehicle-treated control ( Figure 4A) and ampakine-treated ( Figure 4B) mice. Ampakine injection increased ⁇ -gal expression in the CA1 and CA2 of the hippocampus and in the deep layers of the cortex.
  • the invention describes a transgenic mouse which contains an exogenous gene (i.e, a "transgene") in its genome which is propagated in the genome when the animal is reproduced by any means, including by cloning or by the more common sexual crossing to produce progeny.
  • a knockout mouse is a transgenic mouse wherein a preselected allele of the genome has been inactivated by some mechanism, typically by substituting a functional gene with a non-functional gene at that allele by homologous recombination or other mutagenic means.
  • a transgenic mouse having a reporter gene inserted into a region of the N-CAM gene of a chromosomal N-CAM allele of the mouse, wherein the region of insertion is located between a transcription start site and a translation start site in the N-CAM gene, and thereby prevents normal expression of the N-CAM structural gene under the control of the N-CAM promoter. Instead, the inserted reporter gene is expressed under the control of the N-CAM promoter in the knockout allele.
  • the inserted reporter gene is inserted by homologous recombination into the wild-type chromosomal N-CAM allele, and therefore can be engineered to recombine at any of a variety of positions in the N-CAM allele so long as the inserted reporter gene is positioned such that expression of the reporter gene is under the control of the N-CAM promoter.
  • the positioning of the reporter gene into the chromosomal N-CAM allele is a particularly preferred aspect of the invention because it assures that the reporter/knockout construct in the mouse genome is in the native "landscape" of genetic elements of the N-CAM allele.
  • the native landscape is important because it is not well understood how many different genetic elements in the region of the N-CAM allele participate in regulating N-CAM gene expression, nor is it understood how distant from the N-CAM structural gene those elements may be which can participate in the regulation.
  • expression of the reporter gene in the transgenic mouse most accurately reflects as a model the N-CAM promoter activity and regulation of the N-CAM gene when using the invention for screening to identify modulators of N-CAM gene expression because the reporter gene is inserted into the native landscape of the chromosomal N-CAM gene.
  • the inserted reporter gene can be placed at a variety of positions in the N-CAM allele and achieve the desired result, so long as the reporter gene is expressed under the control of the N-CAM promoter.
  • the insertion can be located at the N-CAM allele transcription initiation site, can be located at the N-CAM allele translation initiation site, or at any position in between.
  • An exemplary construct is described in the Examples .
  • the reporter gene expresses a structural gene product which can be detected in the mouse by some means, and a variety of reporter genes are suitable for use in the invention.
  • Exemplary reporter genes include genes which encode the green fluorescent protein (GFP) , the luciferase enzyme, chloramphenicol acetyltransferase, beta galactosidase, and the like proteins. These genes are readily available and the methods for their detection are well known, and therefore, the invention is not to be construed as so limited to any particular reporter gene.
  • the luciferase encoding gene is available in the plasmid pGL3 -Basic from Promega (Madison, Wisconsin); the chloramiphenicol acetyltranserase encoding gene is available in the plasmid pCAT-Basic from Promega; the beta-galactosidase encoding gene is available from many commercial sources on many plasmids as the LacZ gene; and a GFP encoding gene is available on plasmid pEGFP from Clontech (Palo Alto, California) .
  • a preferred transgenic mouse comprises a reporter gene that is the lacZ gene which encodes beta-galactosidase.
  • the lacZ insertion into the N-CAM allele has a structure following homologous recombination using plasmid pNCLKO as described in the Examples and as shown in Figure IB.
  • a beta-galactosidase protein in the context of the present invention refers to a polypeptide which contains beta- galactosidase activity, and is not intended to be restricted to the native bacterial protein.
  • the LacZ gene has been extensively studied and modified in the art, and it is well known that the enzyme can be presented in a variety of forms and retain the enzyme activity, including truncated proteins, fusion proteins, proteins having modified amino acid residue sequences, and the like.
  • the reporter gene can be a fusion protein having multiple functions.
  • One function is the reporter function, ie, to provide a detectable protein by virtue of an activity or binding property, e.g., as an enzyme, an antigen or the like activity.
  • the second function can be any biological activity to supplement the detection system or to supplement the selection procedure.
  • the fusion protein may include a selectable marker such as imparting resistance in cell culture to cytotoxic agents.
  • a preferred selection marker is the protein which imparts resistance to the neo gene and provides selection resistance to G418 as described in the Examples.
  • a preferred embodiment is the fusion protein encoded by the LacZ/GPKneo gene present in plasmid pNCLKO described in the Examples .
  • the present invention is based in part on the ability to decrease or completely suppress the level of expression of N-CAM in the mouse by introducing into the genomic DNA of a mouse a new exogenous DNA sequence that serves to interrupt some portion of the endogenous DNA sequence to be suppressed. Another term for this type of suppression is "knockout".
  • the exogenous nucleic acid is also referred to as a "knockout construct" .
  • the knockout construct in this case the plasmid or expression vector is first prepared and then inserted into an embryonic stem cell in which the construct subsequently becomes integrated into that cells' genomic DNA by the process of homologous recombination.
  • the embryonic stem cell containing the exogenous DNA is then subjected to selection methods as described herein, for example, by selection for neomycin or G418 resistance. Selected resistant cells are then analyzed for the presence of a mutant allele.
  • An embryonic stem cell containing a mutant allele is then injected into a blastocyst that is implanted into the uterus of a pseudopregnant foster mother for integration into a developing embryo .
  • Offspring that are born to the foster mother are then screened for the presence of the mutant allele.
  • a tail sample of the progeny is taken and analyzed by Southern blot hybridization or PCR fragment analysis of genomic DNA using probes specific for the allele, such as is described in the Examples.
  • Animal that contain the N-CAM knockout allele in the germ line are selected and can be used for the generation of homozygous heterozygous animals by sexual crossing.
  • Exemplary teachings of the preparation of disrupted genes and mammals containing such genes are provided in US Patent Nos. 5,553,178, 5,557,032 and 5,569,824, the disclosures of which are hereby incorporated by reference.
  • oligonucleotides and primers are well established in the art, and are useful methods for the production of the reagents to construct the plasmids, primers and transgenic mice described herein. Oligonucleotides and primers can be chemically synthesized, as is well known, and primers can be used in various recombinant DNA methods such as polymerase chain reaction (PCR) to produce copies of genes of interest, such as are described herein, for use in the plasmids and the mice described herein.
  • PCR polymerase chain reaction
  • the invention further contemplates cultured cells which comprise a reporter gene inserted into a region of an N-CAM gene of a chromosomal N-CAM allele of the cell, such that the region of insertion is located between a transcription start site and a translation start site in said N-CAM gene, and thereby place expression of the reporter gene under the control of the N-CAM promoter .
  • the cultured cells can be derived from any tissue of a mouse of the present invention, and therefore can be heterozygous or homozygous for the inserted N-CAM allele according to the invention. That is, tissues and/or cells of a transgenic mouse of this invention can be explanted by any of a variety of methods available in the tissue culture arts to establish a culture of cells, and therefore, the invention is not to be construed as so limited.
  • the explanted cells can be isolated from epithelial or fibroblast tissue, from embryonic, neuronal or endodermal tissue, and the like tissues of the mouse.
  • a preferred tissue is embryonic stem cell tissue, as is described in more detail in the Examples.
  • a cultured cell comprises a reporter gene as described herein for a transgenic mouse, and more preferably is the lacZ gene.
  • a particularly preferred cultured cell line comprises a reporter gene that comprises the nucleotide sequence of the lacZ gene of plasmid pNCLKO described herein that encodes a beta-galactosidase protein.
  • An exemplary cell is the cultured cell line NCL6 having ATCC Accession No. and described further in the Examples.
  • the preparation of a cultured cell line can vary widely, and can include the harvesting of a tissue or cell from the mouse, or from a progeny of a mouse, or from a descendant which is the result of a cross which alters the genotype of the mouse, but maintains the knockout allele of the N-CAM gene as described herein.
  • a cultured cell it is preferred to engineer a cultured cell so that the cell can be readily propagated in tissue culture, and preferably propagated through multiple generation of cell culture and division of the culture.
  • tissue culture preferably propagated through multiple generation of cell culture and division of the culture.
  • the ability to propagate indefinitely is referred to as an immortal cell line, and represents one preferred embodiment of the invention.
  • a preferred method comprises introducing a gene which provides to the cell the ability to propagate in tissue culture indefinitely.
  • the SV40 virus large T antigen is known to promote the ability to grow in culture, and provides one approach towards immortality.
  • the gene which encodes the SV40 large T antigen is known, and can be introduced into the cell to aid in the establishment of a cell line.
  • the gene can be introduced before, during or after explanting the tissue from a mouse, although is most convenient to do so prior to explantation.
  • a preferred method involves engineering a mouse strain which contains a genotype which includes a gene capable of expressing SV40 large T antigen under the control of conditional promoters so that expression of the antigen can be turned on during cell culturing, but can be off in the animal.
  • An exemplary genotype is the "Immortomouse" mouse strain produced by Charles River Laboratories (Wilmington, MA) , in which the SV40 large T antigen is under the control of a H-2Kb promoter that is responsive to gamma interferon, and which expresses the antigen as a temperature sensitive (ts) protein that is functional at 33 degrees Centigrade (33 C) , but is nonfunctional ("off”) at 39 C.
  • This strain has been described in more detail in the Examples .
  • a cell line according to this immortalized embodiment is produced by first crossing a mouse having an N-CAM knockout allele with an Immortomouse, screening for progeny containing both genotypes, and explanting tissues from the progeny to form the cultured cell .
  • the genotype screening for appropriate progeny, and the cell culture conditions to establish the cell culture are described in the Examples .
  • a particularly preferred cultured cell has both the N-CAM knockout allele and the SV40 ts large T antigen, interferon responsive gene.
  • Exemplary is the cell line NCL6 described herein.
  • the invention contemplates methods for in vivo screening of modulators of the N-CAM gene using a transgenic mouse of the present invention having a reporter (i.e., indicator) gene under the expression control of the N-CAM promoter .
  • Potential modulators can be administered to the mouse to characterize the in vivo effect of the modulator upon N-CAM expression.
  • the mouse can be used to show tissue distribution of N-CAM expression under the influence of a putative modulator to be screened by in situ analysis of expression in different tissues.
  • treatments and/or behavior modifications on the mouse can studied for their effect upon the expression of N-CAM.
  • the transgenic mouse can be used as tool for screening for biologically active molecules, treatments, behavior modifications or stimuli which modulate N-CAM expression. For example, by administering a test substance to the mouse and observing expression of the reporter gene, the effect of the substance on N-CAM expression can be identified.
  • the substance can be any type of molecule, composition or therapeutic to be evaluated for the ability to modulate N-CAM expression and can comprise a hormone, a small peptide, synthetic analogs, proteins, complex carbohydrates and the like molecules.
  • a treatment such as a learned or imposed behavior, a physical therapy, a physical stimulus, an electrical or neuro-physiological stimulus or other protocol, when imposed upon the mouse can be evaluated for its effectiveness in modulating N-CAM expression. Combinations of stimuli and chemical substances can also be evaluated.
  • the invention describes a method for screening for a bioactive molecule capable of modulating N-CAM expression comprising the steps of: a) contacting a candidate bioactive molecule with a knockout transgenic mouse according to the present invention; and b) evaluating the expression of the reporter gene in a tissue of the mouse, and thereby the ability of the bioactive molecule to effect expression of N-CAM.
  • the candidate bioactive molecule is typically screened by administration by conventional routes depending upon the class of molecule and the intended therapeutic treatment method.
  • Such methods can include administering the candidate bioactive molecule to the mouse by oral, intravenous, intramuscular, intracranial , subcutaneous, and the like routes.
  • the detection (ie., evaluating) of the expressed reporter gene can be conducted in a variety of methods, and therefore the invention is not to be construed as so limited.
  • detection can be conducted in situ by detecting the expressed protein in a tissue of the mouse, in vitro by harvesting a tissue and measuring the amount of protein therein, or in vivo by imaging the protein in the whole animal or in a whole organ. Detection is also dependent upon the type of reporter gene. Preferred methods of detection are shown in the Examples.
  • transgenic mouse The use of a transgenic mouse according to the present methods is described in the Examples where ampakine is used to modulate the knockout N-CAM promoter and induce the expression of beta-galactosidase.
  • Other candidate bioactive molecules could be similarly screened in the disclosed methods .
  • Cultured cells which contain a reporter gene under the control of the N-CAM promoter can also be used for screening and/or characterizing the effects of potential modulators of the N-CAM promoter.
  • the cultured cell provides particular advantages over the mouse in terms of the degree of difficulty in conducting the screening method both in terms of costs and the manipulative steps for administration of test compounds and screening for reporter gene expression.
  • the invention describes a method for screening for a bioactive molecule capable of modulating N-CAM expression comprising the steps of: a) contacting a candidate bioactive molecule with a cultured mouse cell according to the present invention; and b) evaluating the expression of the reporter gene in the cell, and thereby the ability of the bioactive molecule to effect expression of N-CAM.
  • the contacting step can be conducted by adding compound directly to the tissue culture medium for the cell culture, as is well known, and the cell culture can easily be carried through any culturing conditions as is appropriate for screening. Subsequently, the cells can be treated for evaluation of reporter gene expression.
  • This step can vary widely depending upon the reporter gene and the mechanism for detection of the gene, and can include direct visual inspection following addition of a substrate that reacts with the expressed reporter protein, staining for the expressed protein, immunological detection methods, direct measurement of the protein or protein activity in cell homogenates, and the like methods .
  • a preferred cultured cell is a pluripotent embryonic cell which can be induced to differentiate into a variety of distinctive differentiated tissues, each of which can provide information regarding regulation of the N-CAM promoter.
  • the invention further contemplates a therapeutic method comprising administering to a mammal a composition or compound which modulates N-CAM gene expression.
  • the invention describes an N-CAM modulating compound produced by the screening method of the present invention.
  • the invention describes a composition that modulates N-CAM expression. Therefore the invention describes a method for increasing N-CAM expression comprising administering to a mammal a composition comprising a therapeutically effective amount of a compound which modulates the N-CAM promoter according to the present screening methods .
  • the composition comprises ampakine.
  • Ampakine is a class of compounds well known in the art, and are allosteric modulators of AMPA receptors that enhance normal glutamate-mediated synaptic transmission.
  • a preferred ampakine is CX547.
  • the invention describes a method for modulating N-CAM expression, and neuronal activities mediated by N-CAM expression, comprising administering a therapeutically effective amount of a composition comprising ampakine to a patient exhibiting a condition in which modulation of N-CAM expression is beneficial.
  • mice were produced in which the bacterial lacZ gene was inserted into the 3 ' end of the first exon of the N-CAM gene, thereby disrupting N-CAM expression and placing ⁇ - galactosidase ( ⁇ -gal) expression under the control of the N-CAM promoter.
  • ⁇ -gal ⁇ - galactosidase
  • mice homozygous for the lacZ insertion lacked N-CAM mRNA and protein and showed many of the phenotypic alterations seen in other N-CAM knockout animals, including decreased migration of cells into the olfactory bulb and displacement of pyramidal cells in the CA3 region of the hippocampus.
  • Tomasiewicz et al Neuron . 11:1163-1174 (1993); and Cremer et al , Nature, 367:455-459 (1994).
  • N-CAM deficient animals Methy et al, Neuron , 17:413-422, 1996), they exhibited normal hippocampal LTP.
  • a targeted replacement vector was designed to disrupt the N-CAM allele and to insert a lacZ reporter gene under control of the endogenous N-CAM regulatory sequences ( Figure IB) .
  • the vector contained the 8 kb XhoI-SacII fragment of the N-CAM promoter and the 2 kb Notl-EcoRI fragment as 5 1 and 3' homologous recombinant arms, respectively.
  • coli lacZ gene cassette containing an ATG codon followed by a nuclear localization signal and the neomycin resistance gene driven by the phosphoglycerate kinase (PGK) promoter.
  • PGK phosphoglycerate kinase
  • the lacZ gene was isolated as an Xbal-Hindlll fragment from placF, has a known nucleotide sequence (GenBank accession number V00296) that encodes ⁇ -galactosidase, an enzyme whose activity is readily detected and is not toxic.
  • a PGK-thy ⁇ nidine kinase gene was inserted at the 3 ' end of the N-CAM homologous sequences .
  • the N-CAM genes were obtained from an N-CAM genomic clone which was isolated from the mouse 129/Sv genomic library
  • Figure IB shows the structures of the N-CAM targeting vector, wild-type N-CAM allele, and the disrupted N-CAM allele that was generated after homologous recombination.
  • Restriction enzyme sites are abbreviated as: E, EcoRI ; K, Kpnl; N, NotI; S, Sail; Sa, SacII; X, Xhol. Hatched boxes above the diagram of disrupted N-CAM allele indicate restriction fragments used as probes for Southern blot analysis of the ES cell lines.
  • Probe a is made from the BamHI-Pstl promoter fragment and probe b is made from the Kpnl -Xhol intron 1 fragment of pEC9.7.
  • Plasmid pNCLKO The resulting plasmid, having the features shown in Figure IB, was designated as plasmid pNCLKO, and has been deposited with the ATCC as described herein.
  • restriction digests fragments are diagnostic for the plasmid pNCLKO: Restriction Enzyme Nucleic Acid Fragment Size Xho I 21 kb (linear)
  • transgenic N-CAM knockout mouse was produced having the construct present in plasmid pNCLKO in which the native N-CAM allele is disrupted by homologous recombination to produce a substituted allele which contains the LacZ gene under the expression control the N-CAM promoter.
  • the site of the homologous recombination was between the transcriptional and translational start sites.
  • the E.coli lacZ gene encoding the beta-galactosidase was inserted in the native N-CAM gene ( Figure IB) .
  • Homologous recombination in the 129/Sv ES cell line resulted in a mutant allele with the lacZ reporter gene under control of the transcriptional regulatory regions of the N-CAM gene while at the same time blocking expression of the wild type N-CAM protein.
  • the plasmid pNCLKO was introduced into the embryonic stem cell strain ES-D3 using electroporation to form a transfected cell.
  • 50 ug of plasmid DNA was electroporated onto 7.5 x 10 7 ES-D3 cells, and selection was conducted using G418.
  • the colony forming frequency following electroporation and selection was 5.2 x 10 ⁇ 6 colonies per cell. Twenty five colonies were isolated and were implanted into blastocysts generated from a C57B1/6J mouse for producing a transgenic mouse.
  • ES-D3 cells are an established stem cell culture prepared from pluripotent stem cells of an mouse 129 strain which spontaneously differentiate into embryonic structures in culture. ES-D3 cells are available from ATCC as Accession No. CRL-11632.
  • transfected 129/SV ES cell clones from ES cell clone D selected for G418 resistance, five contained the recombined allele as assessed by Southern blot analysis of Notl/Xhol digests of genomic DNA probed with probe a ( Figure IB; data not shown), including the 10.5 kb fragment indicating homologous recombination to form the disrupted allele.
  • Cells were subjected to karyotype analysis to insure a normal chromosome complement. a single ES cell clone with the identified disrupted allele and a normal karyotype was injected into C57B1/6J blastocysts to generate chimeric founder mice.
  • One of the chimeric animals transmitted the recombinant allele through the germline resulting in animals heterozygous for the N-CAM null allele.
  • the offspring of this animal were used to establish heterozygous and homozygous animals in the background of the inbred C57B1/6J mouse strain and the outbred CD-I strain.
  • a 3' primer was designed (5' CGC CAG GGT TTT CCC AGT CAC GAC G 3') (SEQ ID NO 3) that corresponds to the M13 (-40) sequencing primer located near the polylinker region of the pnLacF vector.
  • This M13 3' primer was used in combination with the above 5 ' primer complementary to the N-CAM promoter to amplify a 305 bp PCR fragment.
  • N-CAM/lacZ knock-out mouse The phenotype of the N-CAM/lacZ knock-out mouse was verified by both northern blot analysis and western blot analysis. Mice homozygous for the disrupted N-CAM allele had no expression of either N-CAM protein (western) or N-CAM mRNA (northern) . a . Northern Blots
  • Northern blot analysis was conducted on the knockout mouse to characterize the expression of mRNA corresponding to the gene for the N-CAM/LacZ construct, and to verify the expression of the wild type allele for N-CAM in wild-type mice or mice heterozygous for N-CAM/LacZ.
  • Fig. IC Northern blot analysis
  • a probe corresponding to base pairs 183 to 505 of the N-CAM cDNA hybridized to RNA from wild type and heterozygous mouse brains but not to RNA from mice homozygous for the N-CAM gene disruption. Ethidium bromide staining indicated that similar amounts of RNA were loaded on the gel (Fig. ID) .
  • N-CAM in the extracts from the wild type and heterozygous mice (Fig. IE) .
  • the N-CAM antibody did not bind to any proteins in the extracts from mice homozygous for the N-CAM gene disruption.
  • Insertion of the bacterial lacZ gene into the first exon and intron of the N-CAM gene disrupted expression of both N-CAM mRNA and protein ( Figure IC, E) .
  • Mice homozygous for the gene disruption expressed no gross phenotypic abnormalities other than a reduced body size and decreased breeding efficiency.
  • the knockout mice described herein have some of the morphological defects observed in other mutants (Tomasiewicz et al, Neuron, 11:1163-1174 (1993); and Cremer et al , Nature, 367:455-459, 1994) although the various N-CAM knockout strains described here and by others represent different alleles due to mutation at widely distributed sites within the N-CAM gene.
  • ⁇ -gal enzymatic assay Two to three millimeter sagittal sections of the hippocampus from each hemisphere were isolated and prepared individually. The tissue was lysed by sonication in 100 mM Tris-acetate, pH 7.8 , 10 mM Mg acetate, 1 mM EDTA, 1% TX-100, 0.2% deoxycholate . Lysate was assayed for ⁇ -gal activity with the FluoReporter kit (Molecular Probes, Eugene, Oregon) according to manufacturer's protocols. ⁇ -gal values were normalized to DNA content of the samples as determined by the Picogreen assay (Molecular Probes) . The values for ⁇ -gal from each hemisphere were averaged. Within an experimental group, ⁇ -gal activity for individual treatments was compared to the average ⁇ -gal levels for control animals. The percent increase was averaged over all experiments and analyzed statistically using the Wilcoxon matched pairs test.
  • ⁇ -gal continued to be expressed throughout the spinal cord and in the dorsal root ganglia (Fig 2C, D) and in heart and kidney (data not shown) and was similar to the pattern of in si tu hybridization for N-CAM mRNA (Fig. 2F) .
  • Fig. 2F dorsal root ganglia
  • Fig. 2G trigeminal ganglia
  • ⁇ -gal expression was increased and was localized to post-mitotic neurons of the midbrain and hindbrain and along the floor plate of the forebrain (Fig.
  • mice Hippocampal morphology in N-CAM deficient mice
  • Homozygous knockout mice had a bifurcation of the CA3 region of the hippocampus not present in wild type animals (Fig. 3A, B) , but similar to that reported in other N-CAM knockout mice (Tomasiewicz et al , Neuron . 11:1163-1174 (1993); and Cremer et al, Mol . Cell. Neurosci.. 8:323-335, 1997).
  • the homozygous mice appeared to have fewer pyramidal cell nuclei in the CA1, CA2 and CA3 regions of the hippocampus.
  • Heterozygous animals showed an intermediate morphological phenotype with more pyramidal neurons and a less pronounced bifurcation.
  • the projections from the dentate gyrus to the CA3 region were similar in mutant and wild type animals as revealed by Timm's staining. It was also apparent in these sections that homozygous animals had an increase in the number of cells in the sub-ventricular zone on the route of migration to the olfactory bulb. They also showed a reduction in the size of the olfactory bulb.
  • the stimulus-driven field excitatory post-synaptic potentials were amplified with an AxoClamp 2B amplifier to a final gain of 1000, low-pass filtered at 2 or 5 kHz, digitized at 5 or 10 kHz, analyzed on-line and then stored on disk for further analysis.
  • the data from an LTP run were considered acceptable if responses to the non-conditioned input deviated in amplitude by less than 20% of initial value.
  • the LTP group statistics were derived from all such runs, regardless of the magnitude of LTP observed. Group statistics were derived after calculating an averaged outcome for each animal; hence, the stated sample sizes are animal counts, rather than the actual number of slices tested (2 to 3 per animal) .
  • Baseline synaptic physiology and LTP was measured in hippocampal slices from heterozygous and homozygous N-CAM knockout mice in comparison with those of wild type animals .
  • fEPSPs field excitatory postsynaptic potentials
  • Paired-pulse facilitation of the fEPSPs was compared in slices from the homozygous mutant and wild type mice, using an interstimulus interval of 25 ms (Table 1 and Fig. 3D) .
  • Mean facilitation ratios of 1.60 + 0.06 and 1.47 ⁇ 0.03 were observed, which did not differ significantly and were similar to levels described in previous studies of these synapses.
  • Half-maximal baseline fEPSPs used for LTP runs were 1 to 2 mV and were elicited by stimulus currents between 20 and 25 ⁇ A.
  • TBS in homozygous mutant and wild type animals elicited a brief ( ⁇ 2 min) post-tetanic potentiation (PTP) , a variable and also brief ( ⁇ 5 min) heterosynaptic depression, short-term potentiation, and LTP, the latter two phenomena being superimposed for the first 20 to 30 min following TBS.
  • PTP post-tetanic potentiation
  • ⁇ 5 min heterosynaptic depression
  • LTP long-term potentiation
  • mice that lacked N-CAM exhibited normal LTP and baseline synaptic physiology.
  • mice that lacked N-CAM exhibited normal LTP and baseline synaptic physiology.
  • mice Heterozygous mice were used to examine regulation of the N-CAM promoter in response to enhanced synaptic transmission since, in these mice, ⁇ -gal expression from the lacZ gene insertion and N-CAM mRNA expression could both be used to monitor the activity of the N-CAM promoter.
  • Synaptic activity was enhanced by treatment with ampakines, a class of drugs that are positive allosteric modulators of AMPA receptors which increase the amplitude and prolong the time course of synaptic responses to endogenous glutamate release.
  • ampakine CX547 a positive allosteric modulator of alpha-amino-3 -hydroxy-5-methyl-4-isoazole propionic acid (AMPA) -type glutamate receptors, was used.
  • AMPA alpha-amino-3 -hydroxy-5-methyl-4-isoazole propionic acid
  • mice Heterozygous mice were injected intraperitoneally (i.p.) with 80 mg/kg of the ampakine CX547 dissolved in 50% PBS with 20% w/v of the carrier cyclodextran. Control animals were injected i.p. with the vehicle alone (20% cyclodextran w/v in 50% PBS) .
  • RNAse protection Eight hours after treatment with ampakine, the brains were harvested, sectioned, and stained histologically for ⁇ -gal activity. Hippocampal tissue was also homogenized and assayed for ⁇ -gal activity as described earlier. After homogenization of individual brains, N-CAM mRNA levels were also measured by RNAse protection as described below.
  • % increase in vi tro Mean treatments 1 2 3 4 5.
  • RNAse protection assays RPAs
  • RNAse protection assays were performed on total brain RNA isolated using RNAzol (Tel-Test, Friendswood, TX) extraction from ampakine- or vehicle-treated animals.
  • RNAzol Tel-Test, Friendswood, TX
  • N-CAM values were normalized to levels of ⁇ -actin mRNA as determined by the protection assay.
  • hippocampal slices were cultured from mice heterozygous for the lacZ insertion into the N-CAM gene and the slices were then exposed to ampakines in vi tro .
  • mice expressing lacZ under the control of the N-CAM promoter showed increased ⁇ -gal reporter activity and N-CAM mRNA levels in response to ampakine treatment.
  • mice heterozygous for the lacZ insertion it is demonstrated in the same animal that there were similar increases in the levels of both N-CAM mRNA and ⁇ -gal activity.
  • CX547 had no effect on fibroblasts cultured from heterozygous embryos and the effects of the drug on tissue slices was reduced by CNQX, a specific antagonist of AMPA receptors.
  • N-CAM mRNA increased in ampakine-treated heterozygous knockout mice and wild type mice as shown by RNAse protection assays (RPA) .
  • RPA RNAse protection assays
  • the response to ampakine was also observed in organotypic slice cultures of the hippocampus. In such slices, the response was similar to that found when glutamate receptors were stimulated with the agonist kainic acid and it was prevented by CNQX, an AMPA receptor antagonist. Together these results indicate that facilitation of AMPA receptors leads to increased N-CAM promoter activity.
  • An established cultured cell line which expresses LacZ under the control of the N-Cam promoter is prepared by crossing in the first generation a male homozygous N-CAM knockout transgenic mouse with a female heterozygous Immortomouse (Charles River Laboratories, Wilmington, MA). The construction and characterization of Immortomouse is described by Jat et al . , Proc . Natl . Acad . Sci . USA. 88:5096-5100 (1991).
  • Immortomouse is a transgenic mouse which contains a gene that expresses the SV-40 large T antigen with a temperature sensitive tsA58 mutation under the control of a gamma interferon-inducible H-2Kb promoter.
  • PCR analysis of genomic DNA isolated from tail samples from the pups was used to identify those cultures made from pups which were heterozygous for the immortomouse gene and either homozygous, heterozygous or wild type for the N-CAM knockout allele .
  • Verification of the Immortomouse allele in progeny is accomplished using PCR of genomic DNA isolated from the tail samples using PCR primers specific for the Immortomouse' s large T antigen expression construct.
  • the primers used are 5 ' -GCA GAA CTA AGA AGT CGC GA-3 ' (SEQ ID NO 4) and 5 ' -GAC ACT CTA TGC CTG TGT GG-3 ' (SEQ ID NO 5), which hybridize to the H-2Kb promoter domain of the large T antigen construct and to the structural protein for the large T antigen. If the Immortomouse allele is present, the PCR fragment is approximately 1000 base pairs.
  • the protocol for genotyping the Immortomouse uses the two primer in a single reaction to detect a specific PCR fragment.
  • the PCR reaction mix contains 400 nanograms (ng) genomic DNA, 2.5 ul DMSO (5% of total reaction volume), 1 ul 10 mM dNTP, 0.25 ul Taq Polymerase (1.25 units), 5 ul 10xMgCl2 Taq Polymerase buffer, 200 ng of each primer, sterile water for a total reaction volume of 50 ul .
  • the PCR reaction was run once at 95 C for 3 min, 30 cycles as follows: 95 C for 1 min, 62 C for 1 min, and 72 C for 1 min; followed by one cycle at 72 C for 8 min.
  • Stem cell culture produced by a heterozygous N-CAM knockout mouse identified by the above PCR genotype analysis was selected and designated as stem cell culture NCL6.
  • NCL6 was deposited with the ATCC as described herein.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Environmental Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention décrit une souris transgénique présentant un allèle N-CAM inactivé avec une insertion de gène marqueur régulé par le promoteur N-CAM. Dans des réalisations préférées, le gène marqueur code pour la bêta-galactosidase. La présente invention comprend aussi des méthodes de détection des modulateurs du gène N-CAM à l'aide de la souris transgénique, ou des cellules cultivées obtenues au moyen de ladite souris.
EP99908469A 1998-02-25 1999-02-25 SOURIS TRANSGENIQUE A GENE MARQUEUR LacZ REGULE PAR LE PROMOTEUR N-CAM SERVANT A L'IDENTIFICATION DE MODULATEURS N-CAM, METHODES ET COMPOSITIONS Withdrawn EP1182924A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US7598598P 1998-02-25 1998-02-25
US75985P 1998-02-25
PCT/US1999/004118 WO1999043783A2 (fr) 1998-02-25 1999-02-25 SOURIS TRANSGENIQUE A GENE MARQUEUR LacZ REGULE PAR LE PROMOTEUR N-CAM SERVANT A L'IDENTIFICATION DE MODULATEURS N-CAM, METHODES ET COMPOSITIONS

Publications (2)

Publication Number Publication Date
EP1182924A2 true EP1182924A2 (fr) 2002-03-06
EP1182924A4 EP1182924A4 (fr) 2003-01-02

Family

ID=22129195

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99908469A Withdrawn EP1182924A4 (fr) 1998-02-25 1999-02-25 SOURIS TRANSGENIQUE A GENE MARQUEUR LacZ REGULE PAR LE PROMOTEUR N-CAM SERVANT A L'IDENTIFICATION DE MODULATEURS N-CAM, METHODES ET COMPOSITIONS

Country Status (2)

Country Link
EP (1) EP1182924A4 (fr)
WO (1) WO1999043783A2 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9925125D0 (en) 1999-10-22 1999-12-22 Novartis Ag Organic compounds
WO2002045495A2 (fr) * 2000-12-06 2002-06-13 Deltagen, Inc. Souris transgeniques contenant disruptions geniques ciblees
US8952213B2 (en) 2002-04-26 2015-02-10 The Board Of Trustees Of The Leland Stanford Junior University Neuronal activation in a transgenic model
US7425618B2 (en) 2002-06-14 2008-09-16 Medimmune, Inc. Stabilized anti-respiratory syncytial virus (RSV) antibody formulations

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5792743A (en) * 1995-04-19 1998-08-11 Acorda Therapeutics Method for promoting neural growth comprising administering a soluble neural cell adhesion molecule

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ARAI A ET AL: "A CENTRALLY ACTIVE DRUG THAT MODULATES AMPA RECEPTOR GATED CURRENTS" BRAIN RESEARCH, AMSTERDAM, NL, vol. 638, 1994, pages 343-346, XP000905167 ISSN: 0006-8993 *
HOLST BRENT D ET AL: "A binding site for Pax proteins regulates expression of the gene for the neural cell adhesion molecule in the embryonic spinal cord." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 94, no. 4, 1997, pages 1465-1470, XP002217714 1997 ISSN: 0027-8424 *
See also references of WO9943783A2 *
WANG YIBIN ET AL: "Embryonic expression patterns of the neural cell adhesion molecule gene are regulated by homeodomain binding sites." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 93, no. 5, 1996, pages 1892-1896, XP002217713 1996 ISSN: 0027-8424 *

Also Published As

Publication number Publication date
EP1182924A4 (fr) 2003-01-02
WO1999043783A3 (fr) 2001-12-13
WO1999043783A2 (fr) 1999-09-02

Similar Documents

Publication Publication Date Title
Zhang et al. A selective defect in IgG2b switching as a result of targeted mutation of the I gamma 2b promoter and exon.
Matsumoto et al. Ataxia and epileptic seizures in mice lacking type 1 inositol 1, 4, 5-trisphosphate receptor
Li et al. Whisker-related neuronal patterns fail to develop in the trigeminal brainstem nuclei of NMDAR1 knockout mice
Lüthi et al. Endogenous serine protease inhibitor modulates epileptic activity and hippocampal long-term potentiation
Zhang et al. Highly restricted expression of Cre recombinase in cerebellar Purkinje cells
JP3679403B2 (ja) トランスジェニック動物
Yoon et al. DifferentMRF4Knockout Alleles Differentially Disrupt Myf-5 Expression: cis-Regulatory Interactions at theMRF4/Myf-5Locus
US20130071407A1 (en) Methods for modulating hematopoiesis and vascular growth
US5814300A (en) Gene-targeted non-human mammals deficient in the SOD-1 gene
EP1182924A2 (fr) SOURIS TRANSGENIQUE A GENE MARQUEUR LacZ REGULE PAR LE PROMOTEUR N-CAM SERVANT A L'IDENTIFICATION DE MODULATEURS N-CAM, METHODES ET COMPOSITIONS
AU732477B2 (en) Ku deficient cells and non-human transgenic animals
JP4450874B2 (ja) ニューロンニコチン性アセチルコリン受容体のβ２−サブユニットの調節配列及びこれをコードする配列を含むゲノムＤＮＡ断片、及びこれらの断片又は突然変異断片を用いて作製されたトランスジェニック動物
US20020104107A1 (en) Point mutant mice with hypersensitive alpha 4 nicotinic receptors: dompaminergic pathology and increased anxiety
EP0984070A1 (fr) Procédé pour détecter des substances pour la thérapie des maladies associées avec apoptosis transmisé par c-Jun
US20120095084A1 (en) Modulation of serotonin neurons involving the pet-1 transcriptional control region
CA2418267C (fr) Procede de criblage de l'activite de promoteurs ou d'inhibiteurs du ptp zeta
AU781591B2 (en) Compositions and methods for improving learning and memory
Balu et al. Behavioral and physiological characterization of PKC-dependent phosphorylation in the Grin2a∆ PKC mouse
JP2005502341A (ja) Ｂ１ブラジキニン受容体タンパク質を調節するための動物モデルとしてのトランスジェニックげっ歯類
US6822138B1 (en) NIK-knockout mice
EP1619248B1 (fr) Souris presentant une deficience de la fonction glast de transporteur de glutamate
EP0647395A2 (fr) Souris déficientes en récepteurs de la neurotrophine
CA2447276C (fr) Modele animal non humain du trouble du developpement des oligodendrocytes
KR100514090B1 (ko) Ｎｃｘ2 단백질의 활성을 억제함으로써 학습능력 및기억력을 증진시키는 방법
JP2003018944A (ja) 細胞死誘導モデル非ヒト動物

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000830

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

RIC1 Information provided on ipc code assigned before grant

Free format text: 7A 01K 67/00 A, 7G 01N 33/00 B, 7A 61K 49/00 B, 7C 12N 15/63 B, 7C 12N 15/85 B, 7A 01K 67/027 B, 7C 07K 14/705 B

A4 Supplementary search report drawn up and despatched

Effective date: 20021111

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20030124