WO2006022457A1 - Synoviolin transgenic flies - Google Patents

Synoviolin transgenic flies Download PDF

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Publication number
WO2006022457A1
WO2006022457A1 PCT/JP2005/016072 JP2005016072W WO2006022457A1 WO 2006022457 A1 WO2006022457 A1 WO 2006022457A1 JP 2005016072 W JP2005016072 W JP 2005016072W WO 2006022457 A1 WO2006022457 A1 WO 2006022457A1
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fly
phenotype
wing
dsyno
transgenic
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PCT/JP2005/016072
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English (en)
French (fr)
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Toshihiro Nakajima
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Locomogene, Inc.
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Priority to JP2007509812A priority Critical patent/JP2008510451A/ja
Publication of WO2006022457A1 publication Critical patent/WO2006022457A1/en

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • 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
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0337—Genetically modified Arthropods
    • A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases

Definitions

  • the present invention relates to a transgenic fly comprising a gene encoding the Synoviolin.
  • RA Rheumatoid arthritis
  • RA RA-associated regional pain syndrome
  • TNF ⁇ tumor necrosis factor ⁇
  • IL interleukin-I
  • IL-6 IL-6
  • cytokines stimulate the overgrowth of synovial cells to form a mass of synovial tissue, called pannus, which invades the bone and cartilage through osteoclast activation and protease production (Tak and Bresnihan 2000; Hofbauer and Heufelder 2001; Kaneko et al. 2001; Rehman and Lane 2001; Szekanecz and Koch 2001).
  • RA is considered an autoimmune disease
  • medical treatment targeting inflammation has been applied.
  • TNF ⁇ blockade therapy hardly leads to complete remission of the disease and approximately 25 percent of the patients with RA do not respond to such therapy (Green 2000; Clair 2002).
  • the pathogenesis of RA cannot be explained in all patients by inflammation only. In other words, it is not clear whether inflammation causes subsequent synovial hyperplasia or whether primary spontaneous synovial cell proliferation leads to inflammation. In any case, there is a general agreement that synovial cells play an important role in RA.
  • Synoviolin has unexpectedly inhibits p53 activity in cultured mammalian cells. Despite intensive efforts, the complex p53 network in mammalian cells is far from fully understood. Moreover, the majority of this information obtained from cultured cells may not fully reflect the physiological function of p53 in the context of the whole organism. DISCLOSURE OF INVENTION
  • the present inventors have succeeded in establishing a transgenic fly.
  • the inventors applied fly-genetics approaches and first confirmed that dsyno is able to ubiquitinate Drosophila homolog of p53 (Dmp53) in vi ⁇ ro.
  • the present invention is as described below.
  • the transgenic fly of (1) wherein the fly displays inhibition of transportation of p53 to a nuclear in a cell.
  • a method for expressing Synoviolin in a fly comprising introducing a gene encoding the Synoviolin into a genome of the fly.
  • a method for screening a compound having an activity to inhibit ' Synoviolin expression comprising:
  • a method for screening a compound useful for anti-tumor drug comprising:
  • a method for analyzing a regulatory function of p53 in target cells or tissues comprising the steps of:
  • a method for screening a compound having an activity to enhance apoptosis or cell proliferation comprising:
  • a method for screening a compound having an activity to inhibit apoptosis or cell proliferation comprising:
  • the dominant negative form of the transgenic fly of (1) comprises a polypeptide of which cystein at the 305* amino acid residue as shown in SEQ ID NO: 2 is substituted with serine.
  • Figure 1 shows a result of the in vitro auto-ubiquitination assay.
  • Figure 2 shows an effect of CG1937 overexpression in UAS-dsyno (GHl 1117) transgenic fly with tissue-specific Gal4 driver.
  • Figure 3 shows a result of an immunostaining of Drosophila imaginal discs with anti-Syno antibody.
  • Figure 4 shows a result of in situ hybridization of CGl 937 in different stages of Drosophila embryos.
  • Figure 5 shows a result of in situ hybridization of CG 1937 in imaginal discs at
  • Figure 6 shows a result of expression of CG1937 at different developmental stage of Drosophila (RT-PCR).
  • Figure 7 shows two EP lines in UTR region of CGl 937.
  • Figure 8 shows a scheme for generation of dsyno deletion mutant.
  • Figure 9 shows a result of expression of CG1937 (dsyno) in GE27750 and GE27750(R).
  • Figure 10 shows an effect of CG1937 overexpression in GE27750 with tissue-specific Gal4 driver.
  • Figure 11 shows an eye phenotype of gmr-Gal4; UAS-dsyno transgenic lines
  • Figure 12 shows a wing phenotype of Ser-Gal4; UAS-dsyno transgenic fly.
  • Figure 13 shows an eye phenotype of gmr-Gal4; UAS-dsyno transgenic lines (29 0 C).
  • Figure 14 shows a wing phenotype of MS1096-Gal4; UAS-dsyno transgenic fly.
  • Figure 15 shows a wing phenotype of el6E-Gal4; UAS-dsyno transgenic fly.
  • Figure 16 shows an effect of CGl 937 overexpression in UAS-dsyno transgenic fly with el6E-Gal4 driver.
  • Figure 17 shows an expression of CGl 937 in UAS-dsyno transgenic fly (RT-PCR).
  • Figure 18 shows an annotation of UAS-dsyno transgenic lines by Inverse PCR.
  • Figure 19 shows larval wing discs of MS1096-Gal4; wg-LacZ; UAS-dsyno fly.
  • Figure 20 shows an effect of dominant negative form of CGl 937 overexpression with tissue-specific Gal4 driver.
  • Figure 21 shows wing defects of el6E-Gal4; UAS-dsyno DN#12 transgenic lines.
  • Figure 22 shows a wing phenotype of Ser-Gal4; UAS- dsyno ⁇ 12 fly.
  • Figure 23 shows an eye phenotype of gmr-Gal4; UAS-dsyno ⁇ 72 at 29 0 C.
  • Figure 24 shows an expression of dominant negative form of CG1937 (C305S) in UAS-dsyno DN transgenic fly (RT-PCR).
  • Figure 25 shows mitotic clones of GE27750 in wing disc.
  • Figure 26 shows mitotic clones of GE27750 in adult eye.
  • Figure 27 shows a wing phenotype of el 6E-Gal4; UAS- Dmp53 transgenic fly.
  • Figure 28 shows a wing phenotype of el6E-Gal4; UAS- Dmp53 transgenic fly.
  • Figure 29 shows an immunostaining of a wing phenotype of el6E-Gal4; UAS- Dmp53 transgenic fly with anti-p53 antibody.
  • Figure 30 shows an acridine orange staining of el6E-Gal4; UAS- Dmp53 transgenic fly.
  • the present invention relates to a transgenic fly comprising a gene encoding the Drosophila Syno violin (dsyno).
  • the overexpression of dsyno causes visible phenotype in wings and eyes.
  • overexpression of dominant negative form of dsyno (C305S) does not cause any visible phenotype in a fly. Therefore, the transgenic fly of the present invention may be used as a model fly for screening of dsyno modifier.
  • the transgenic fly comprising a gene encoding dsyno may be used as a model fly for screening of a compound useful for an anti-cancer drug.
  • transgenic organism refers to an organism that has had extra genetic material inserted into its genome.
  • a "transgenic fly” includes embryonic, larval and adult forms of Drosophila melanogaster that contain a DNA sequence from the same or another organism randomly inserted into their genome. Although Drosophila melanogaster is preferred, it is contemplated that any fly of the genus Drosophila may be used in the present invention.
  • transgene refers to expression of the transgene in a tissue or cell or at a specific developmental stage where it is not normally expressed.
  • phenotype refers to the observable physical or biochemical characteristics of an organism as determined by both genetic makeup and environmental influences.
  • promoter refers to DNA sequences which are recognized directly or indirectly and bound by a DNA-dependent RNA polymerase during the initiation of transcription and includes enhancer elements. Enhancers used in the present invention include the UAS element which is activated by the yeast Gal4 transcriptional regulator. "UAS” element as used herein refers to an upstream activating sequence recognized by the GAL-4 transcriptional activator.
  • transcription factor refers to any protein required to initiate or regulate transcription in eukaryotes.
  • a "control" fly refers to a larva or fly that is of the same genotype as larvae or a fly used in the methods of the present invention except that the control larva or fly does not carry the mutation being tested for modification of phenotype, or is not administered candidate compounds.
  • the Synoviolin used in the present invention is an E3 ubiquitin ligase and found in synovial cells in RA patients.
  • the Synoviolin activity of the present invention means an auto-ubiquitination activity.
  • the Synoviolin is highly conserved from yeast to human.
  • Drosophila Synoviolin (dsyno) may be used.
  • dsyno gene derived from Drosophila melanogaster, CGl 937 may be used.
  • Drosophila Synoviolin (dsyno) gene, CGl 937 is a fly homolog of mammalian Synoviolin which has the highest homology (63%) with mammalian Synoviolin.
  • the dsyno gene may be obtained from genome library of fly.
  • the desired clone may be obtained from bacterial artificial chromosome (BAC) library by hybridization method and be obtained by PCR method or more.
  • the Drosophila Synoviolin (dsyno) gene may be any DNA hybridizable a complementary DNA to a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 under highly stringent conditions and has a Synoviolin activity.
  • the hybridization can be carried out by publicly known methods or by modifications of these methods, for example, according to the method described in Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989).
  • the highly stringent conditions used herein are, for example, those in a 1-2 X SSC and 0.1 - 0.5 % SDS at a temperature of about 48 to 68°C. 3.
  • the present invention relates to a transgenic fly comprising a gene encoding the Synoviolin.
  • the fly that can be used include the members of the family Drosophilidae, preferably, Drosophila melanogaster.
  • the present invention discloses a transgenic fly that contains a DNA sequence (SEQ ID NO: 1) encoding a polypeptide (SEQ ID NO: 2) comprising the Synoviolin in its genome.
  • the DNA sequences are operably linked to tissue-specific expression control sequences such as promoter regions or upstream activating sequences (UAS), depending on the expression system utilized. These expression control sequences include those that are specific for organs such as eye, wing, leg and haltere.
  • tissue specific control sequences Under the control of these tissue specific control sequences, encoded peptides are transcribed to form mRNA which is translated into detectable levels of dsyno peptide and which causes altered phenotypes in the flies. By assaying for changes in these phenotypes, these flies can be used to identify genes or compounds that may affect the inhibition pathway of Synoviolin and may provide insight into the molecular and biochemical mechanisms of the interaction between p53 and Synoviolin.
  • transgenic fly including transgenic Drosophila
  • P-element mediated transformation is Spradling, 1986.
  • the EP element technology refers to a binary system, utilizing the yeast Gal4 transcriptional activator and UAS sequences, which is used to ectopically regulate the transcription of endogenous Drosophila genes. This technology is described in: Brand and Perrimon, 1993.
  • Conventional expression control systems may be used to achieve ectopic expression of proteins of interest, including the dsyno peptides of the present invention. Such expression may result in the disturbance of biochemical pathways and the generation of altered phenotypes.
  • One such expression control system involves direct fusion of the DNA sequence to expression control sequences of tissue-specifically expressed genes, such as promoters or enhancers.
  • Another expression control system that may be used is the binary Gal4-transcriptional activation system.
  • the Gal4 system uses the yeast transcriptional activator Gal4, to drive the expression of a gene of interest in a tissue specific manner.
  • the Gal4 gene has been randomly inserted into the fly genome, using a conventional transformation system, so that it has been under the control of genomic enhancers that drive expression in a temporal and tissue-specific manner. Individual strains of flies have been established, called “drivers", which carry those insertions.
  • a gene of interest is cloned into a transformation vector, so that its transcription is under the control of the UAS sequence (Upstream Activating
  • the Gal4-responsive element When a fly strain that carries the UAS-gene of interest sequence is crossed to a fly strain that expresses the Gal4 gene under the control of a tissue specific enhancer, the gene will be expressed in a tissue specific pattern.
  • Gal4 "drivers” that drive expression in later stages of the fly development may be used in the present invention. Using these drivers, expression would result in possible defects in the wings, the eyes, the legs, and different sensory organs.
  • These "drivers” include, for example, Ser-Gal4 (wings), MS1096-Gal4 (wings), el6E-Gal4 (wings), gmr-Gal4(eyes) and pGMR-Gal4 (eyes).
  • Various DNA constructs may be used to generate the transgenic Drosophila melanogaster of the present invention.
  • the construct may contain the
  • Synoviolin gene and operably linked to the tissue-specific promoter. Insertion of these constructs into the fly genome may occur through P-element recombination, homologous recombination or other standard techniques known to one of skill in the art.
  • the transgenic fly of the present invention is prepared by introducing a recombinant vector into embryos.
  • the recombinant vector is prepared by sub-cloning a desired DNA into an expression vector.
  • the recombinant vector may be obtained by conventional genetic engineering techniques. Synoviolin gene may be amplified by carrying out RT-PCR and/or DNA-PCR using adequate primers.
  • Synoviolin DNA fragments may be cut out using a known restriction enzyme. Subsequently, the Synoviolin fragment may be inserted into a known vector and then an adequate promoter sequence cut out with restriction enzymes may be inserted upstream of
  • Drosophila-de ⁇ ved plasmids As expression vectors for the Synoviolin, Drosophila-de ⁇ ved plasmids, Escherichia co/z-derived plasmids, Bacillus subtilis-de ⁇ ved plasmids, yeast-derived plasmids, or more may be used. Drosophila-de ⁇ ved plasmids may be preferably used. Examples of Drosophila-de ⁇ ved plasmids include pUAST, pUAS, pChs and pPTGAL.
  • the DNA transgenic fly of the present invention can be prepared using any conventional protocol.
  • a numbers of different strategies can be employed to obtain the integration of the synoviolin DNA (transgene) with the requisite expression pattern.
  • methods of producing the subject transgenic fly involve stable integration of the transgene which is present on a suitable vector, such as a plasmid into a cell or cells of the fly, e.g. germ cell of embryo.
  • Transgene introduction may be accomplished using any conventional protocols. Suitable protocol is well-known to those skilled in the art, such as the microinjection (Spradling, A.C. (1986)).
  • UAS-dsyno transgenic (TG) fly may be prepared by sub-cloning GHl 1117 which is an EST clone of CGl 937 into the pUAST vector.
  • the GHl 1117 may be purchased (Open Biosystems).
  • the resulting UAS-dsyno vector may be introduced into w 1118 fly embryos through microinjection.
  • UAS-dominant negative form of dsyno (dsyno DN ) fly (UAS-dsyno DN fly) may be prepared by a conventional method.
  • CGl 937 cDNA may be modified into CG1937(C305S) that could produce a dominant negative form of dsyno protein and this modified cDNA may be subcloned into pUAS vector.
  • C305S means modified amino acid sequence that cystein at the 305 4 amino acid residue of the CGl 937 polypeptide (SEQ ID NO: 2) is substituted with serine.
  • the pUAS-dsyno DN vector may also be
  • dsyno or dsyno DN may be driven in the various tissues via adequate systems, such as the Gal4-UAS system using the Gal4 drivers containing the various tissue-specific promoters and enhancers.
  • Gal4 drivers gmr-Gal4, Ser-Gal4, MS1096-Gal4, and el 6E-GaU may be preferably used.
  • the expression of dsyno or dsyno DN may be confirmed by immunostaining.
  • Embryos may be fixed as described by Ponzielli et al. (2002).
  • Dissected 3 rd instar larvae may be fixed as described by Solano et al. (2003).
  • In situ hybridization may be performed as described by Ponzielli et al. (2002), for example, using digoxigenin (Dig)-labeled sense or antisense RNA probes specific for CGl 937, except that the linearized DNA templates for synthesis of RNA probes are prepared by RT-PCR from the total RNA of an adult fly, using adequate two sets of primers.
  • transcripts of dsyno are detected in very early stage of embryogenesis or in imaginal discs at 3 r instar larval stages in the fly.
  • the message is continuously expressed throughout embryogenesis in a ubiquitous pattern.
  • the expression of dsyno at developmental stages of Drosophila may be confirmed by investigating expression of dsyno mRNA by RT-PCR.
  • the transgenic fly may be crossed to adequate lines.
  • the progenies are incubated under an adequate condition in third instar larva stage.
  • the total RNA is extracted from the larvae using, for example, easy- Blue Total RNA Extraction kit (iNtRON) according to the manufacturer's instruction. It is used in cDNA synthesis reactions performed with Reverse Transcription system (Promega).
  • the amounts of dsyno and ubiquitously expressed Drosophila ribosomal protein gene rp49 mRNAs may be measured by PCR and gel electrophoresis analysis with adequate primers.
  • the dsyno message may be detected at different developmental stages of a fly from embryo to adult. It is shown that the dsyno (CGl 937) ubiquitously expressed throughout all developmental stages of Drosophila melanogaster.
  • dsyno or dsyno DN transgenic fly causes visible defective phenotype in various tissues, especially, eyes and wings.
  • the expression of dsyno in eyes may cause decolorization or roughness of eye, or glazed eye.
  • the expression of dsyno DN in eyes may not cause any visible phenotypes.
  • the term "decolorization" phenotype is characterized by disorganization of ommatidia and inter-ommatidial bristles and can be caused by degeneration of pigment cells.
  • the term "rough eye" phenotype is characterized by disorganization of ommatidia and inter-ommatidial bristles and can be caused by degeneration of neuronal cells.
  • glazed eyes phenotype is characterized by that the surface of eyes were shinny and smooth as if they are glazed with wax and can be caused by abnormal development of ommatidia. This phenotype is visible through a dissecting stereo-microscope.
  • the expression of dsyno in the wing blade displays a wing defect phenotype, such as a wrinkled wing, bended wing, notching and appearance of a pouch(s) in a wing.
  • the term "wrinkled wing" phenotype is characterized by abnormal folding of the fly wing such that the wings are shrunk. . ⁇
  • the term "bended wing" phenotype is characterized by abnormal folding of the fly wing such that the wings are bent upwards or downwards along their long margins.
  • notching phenotype is characterized by abnormal cut of the fly wing margin such that the wings are serrated along their margins.
  • the term "pouch" phenotype is characterized by abnormal morphology of the fly wing such that a bubble is generated in wings.
  • dsyno is a predominant factor for Dmp53 since the dsyno ubiquitinates Dorosophila homolog of p53 (Dmp53) and metabolizes Dmp53 physiologically.
  • the transgenic fly over-expressing Dmp53 may be generated by using el6E-Gal4 driver.
  • a transgenic fly containing UAS-dsyno and UAS-Dmp53 may be generated by injection of recombinant DNA into Drosophila inelanogaster embryos according to standard procedures (Spradling, 1986).
  • the expression of dsyno and Dmp53 may be driven in the posterior half of the wing using the el6E-Gal4 drivers containing the tissue-specific promoters and enhancers.
  • the el6E-Gal4/UAS-Dmp53; UAS-dsyno/+ fly may be generated from the cross between el6E-Gal4/UAS-Dmp53 females and el6E-Gal4/CyO; UAS-dsyno/TM2 males. Further, each transgenic line may be interbred. As a result, only the bubbled wing phenotypes of UAS-Dmp53 fly is suppressed by overexpression of dsyno in fly (Fig. 28).
  • Dmp53 is remarkably decreased by overexpression of dsyno (Fig. 29).
  • Expression level of Dmp53 in the wing disk of el6E-Gal4/UAS-Dmp53; UAS-dsyno/+ fly may be analyzed to verify dsyno metabolize Dmp53 in vivo by immunostaining as described below.
  • Wing disc may be dissected in PBS, fixed in an adequate buffer, and blocked with an adequate blocking buffer.
  • the fixed wing disk may be incubated under the adequate condition in rabbit anti-Dmp53 antibody. After washing in an adequate wash buffer, they may be incubated under the adequate condition in donkey anti-rabbit FITC, washed with the wash buffer, and then mounted in adequate mounting solutions.
  • the Syno violin involves in physiological regulation of p53 both in fly and mammalian. The regulation of p53 can be confirmed by the acridine orange staining.
  • an ectopically expressed gene may result in an altered phenotype by disruption of a dsyno pathway. Mutations in genes acting in the same dsyno pathway are expected to cause modification of the altered phenotype.
  • this system is extremely beneficial for the elucidation of the function of processed dsyno gene products, as well as the identification of other genes that directly or indirectly interact with them. It is contemplated herein that genes involved in the dsyno pathway can be identified in this manner and these genes can then serve as targets for the development of therapeutics to treat conditions associated with abnormalities in the Synoviolin pathway, leading to diseases, including but not limited to cancer.
  • Said method may comprise contacting candidate compounds to transgenic flies of the present invention and then assaying for changes in the phenotype of the transgenic flies as compared to the phenotype of control transgenic flies that have not been contacted the compound.
  • candidate compounds can be fed to larvae expressing a dsyno. The larvae can then be grown to the adult stage and modification of the dsyno-induced phenotype assayed.
  • Candidate compounds may also be fed to adult flies and modifications of phenotype assayed.
  • test compound for example, oral administration, microinjection or the like are applied, and a necessary treatment may be appropriately selected depending on conditions of the test fly, properties of the test compound, and the like. Furthermore, a dose of the test compound to be administered may be appropriately chosen depending on the administration route, property of the test compound, and the like.
  • the mechanism of action of compounds thus identified may be examined by comparing the phenotypes produced by genetic manipulation with those induced by the administration of a compound of interest. Such compounds include those that may ameliorate or worsen the altered phenotype created in the transgenic flies. Expression of a compound-induced phenotype similar to one associated with a known genetic modification would suggest that the compound has an effect on the same pathway that the genetic modification is affecting.
  • the test compound when a test compound is administered to a test fly and one of phenotypes described above is displayed in the test fly compared to control fly, the test compound can be selected as the compound having an activity of synoviolin.
  • a wide variety of small molecular weight compounds may be used in the screen. Such compounds include, but are not limited to, any compositions which are being tested for lead drug discovery or development. Compounds may be aqueous- or lipid-soluble. Compounds may be dissolved or suspended within solution. Dosages and volumes which are microinjected into the Drosophila may be varied so as to optimize dosages for further studies or to rank compounds as to their toxicity and/or potency.
  • candidate compound examples include genes, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, blood plasma, and the like. These compounds may be novel compounds or publicly known compounds.
  • the compound obtained by the screening method above may form salts, and may be used in the form of salts with physiologically acceptable acids (e.g., inorganic acids, organic acids, etc.) or bases (e.g., alkali metal salts), preferably in the form of physiologically acceptable acid addition salts.
  • physiologically acceptable acids e.g., inorganic acids, organic acids, etc.
  • bases e.g., alkali metal salts
  • in vitro and other in vivo models of diseases For example, numerous cell lines may be used as in vitro models of tumor and are familiar to one of skill in the art, including, for example, the cell lines CCL-116.
  • In vivo models also exist and include, for example, a mammal (e.g. human, mouse or rat) model of diseases.
  • diseases include, but not limited to rheumatoid arthritis, cancer, fibrosis, artheriosclerosis, Castleman's desease, multiple myeloma, Crohn desease, juveile idiopathic systemic arthritis, blain neoplasm, lingual cancer, pharyngeal cancer, pulmonaly carcinoma, mammary carcinoma, esophageal cancer, gastric cancer, pancrea cancer, biliary tract cancer, gallbladder cancer, duodenal cancer, colon cancer, hepatic carcinoma, uterine cancer, ovary cancer, testicular cancer, prostatic cancer, kidney cancer, bladder cancer, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, skin cancer, spleen cancer, thyroid cancer, acute myeloid leukemia, acute lymphoblastic leukemia, adult T-cell leukemia or malignant lymphoma.
  • Drosophila homologs of p53 may be useful to treat tumor or other conditions associated with defects in the regulation of the p53 pathway.
  • dsyno is a predominant factor for Dmp53 since the dsyno ubiquitinates Drosophila homolog of p53 (Dmp53), metabolizes Dmp53 physiologically. Further, Dmp53 is remarkably decreased by overexpression of dsyno. Moreover, the apoptotic cells which are found where Dmp53 is over-expressed are remarkably reduced by over-expressing of dsyno. Therefore, the Syno violin involves in physiological regulation of p53 both in fly and mammalian.
  • the .present invention provides that a method for analyzing a regulatory function of p53 in target cells or tissues.
  • the method comprises the following steps of:
  • p53 By assaying different kinds of biological and/or pathological signal of p53, one can assess the functional aspects of p53 as it relates to proliferation of tumor cells.
  • Certain biological and/or pathological system is particularly well-adapted to exploit this approach.
  • the developmental biology of Drosophila is well characterized, easily reproduced, and suitable for large scale screening methods.
  • examples of biological systems include, but not to be limited to apoptosis and cell proliferation.
  • Gene expression monitoring can be used to establish developmental stages, for example, of a differentiating tissue, of an embryo, or of a larva.
  • a distinctive pattern of gene expression can be established for a particular stage by comparisons to closely and/or distantly related stages. Such patterns can be used for developmental staging of cells or tissues.
  • CG1937 As a Drosophila Synoviolin (dsyno) gene, CG1937 was used. CG1937 was found as the gene which has the highest homology (63%) with mammalian Synoviolin, through blastp (protein-protein blast) in flybase blast site (http://flybase.bio.indiana.edu/blast/). Moreover, the putative RING domain of CGl 937, which is an active center of the ubiquitin ligase, is highly conserved (82%). Therefore, this gene was predicted to fly homolog of mammalian Synoviolin, and could function as an E3 ubiquitin ligase.
  • GST-dsyno ⁇ TM showed the auto-ubiquitination activity same as mammalian Synoviolin (Amano et al., 2003) (Fig.l), and this activity was completely disappeared by the mutation in the RING domain (C305S) of dsyno (Fig.1).
  • CGl 937 is fly homolog of mammalian Synoviolin.
  • EST GHl 1117 containing cDNA of CGl 937 was purchased from Open Biosystems. 2.3 kb ( ⁇ 2282bp) cDNA of CGl 937 ⁇ Drosophila) was cut out from the EST GHl 1117 with EcoRI/Xho I, and subcloned into pUAST vector (Brand and Perrimon, 1993).
  • UAS-dsyno TG fly Transformation of Drosophila melanogaster
  • UAS-dsyno TG fly was generated by injection of recombinant DNA at a concentration of 1 ⁇ g/ ⁇ 1 into Drosophila melanogaster embryos of the genotype W 1118 according to standard procedures well-known to those skilled in the art (Spradling, Drosophila: A Practical Approach, D.B. Roberts, ed., IRL Press, DC(1986), pp. 175-197).
  • UAS-dsyno TG fly an EST clone of CGl 937, GHl 1117, was purchased and subcloned into the pUAST vector. The resulting UAS-dsyno vector was introduced into w 1118 fly embryos through microinjection. 40 transgenic lines were recovered and stabilized (Fig.2).
  • dsyno DN dsyno DN fly
  • UAS-dsyno DN fly UAS-dominant negative form of dsyno (dsyno DN ) fly (UAS-dsyno DN fly)
  • the inventor modified CG1937 cDNA into CG1937(C305S) that could produce a dominant negative form of dsyno protein and this modified cDNA was subcloned into pUAS vector.
  • the pUAS-dsyno DN vector was injected into w 1118 fly embryos. 30 transgenic lines were recovered and stabilized. 3) Expression of synoviolin in Drosophila The expression of dsyno was confirmed by immunostaining as described below.
  • Imaginal discs were dissected in PBS, fixed in a fix buffer of 50 mM Tris-Cl [pH6.8], 1 mM EGTA, 1% Triton X-100, 2 mM MgSO 4 , 150 mM NaCl, 2.2% formaldehyde for 15 minutes, and blocked using a blocking buffer of 50 mM Tris-Cl [pH 6.8], 150 mM NaCl, 0.5% NP40, and 5 mg/ml BSA. The fixed imaginal discs were incubated overnight at 4°C in a 1 : 200 dilution of rabbit anti-Synoviolin antibody.
  • dsyno was also confirmed by in situ hybridization as described below. Embryos were fixed as described by Ponzielli et al. (2002). Dissected 3 rd instar larvae were fixed as described by Solano et al. (2003). In situ hybridization was performed as described by Ponzielli et al. (2002), using digoxigenin (Dig)-labeled sense or antisense RNA probes specific for CGl 937, except that the linearized DNA templates for synthesis of RNA probes were prepared by RT-PCR from the total RNA of an adult fly, using two sets of primers:
  • Synoviolin-AntiSense-Rev 5'-catcatgcagctgctcttatcgtc -3' (SEQ ID NO: 3) and Syno violin- AntiSense-For-T7, 5' -taatacgactcactataggggttgacatcagattgtagggcgtc -3' (SEQ ID NO: 4) 2) systhesis of sense RNA probes;
  • Synoviolin-Sense-Rev 5' -gttgacatca gattgtagggcgtc -3' (SEQ ID NO: 5) and Synoviolin-Sense-For-T7, 5 ' -taatacgactcactatagggcatcatgcagctgctcttatcgtc -3 ' (SEQ ID NO: 6)
  • dsyno The result of the in situ hybridization of dsyno (CGl 937) in different stages of Drosophila embryos was shown in Fig.4. Transcripts of dsyno were detected in very early stage of embryogenesis in a fly. The message was continuously expressed throughout embryogenesis in a ubiquitous pattern.
  • the result of the in situ hybridization of dsyno in imaginal discs at 3 rd instar larval stages was shown in Fig.5.
  • the dsyno transcripts were detected in imaginal discs of 3 rd instar larvae. Eye, antenna, leg, wing discs were stained with an antisense probe of CG1937. It is also detected in larval brain. The staining is not non-specific since the corresponding sense probe did not stain any of these tissues.
  • dsyno at developmental stages of Drosophila was confirmed by investigating expression of dsyno rriRNA by RT-PCR as described below.
  • the transgenic fly was crossed to hsp70-Gal4 lines.
  • the progenies were incubated at 37 0 C for 2hours in third instar larva stage.
  • the total RNA was extracted from the five larvae using easy- Blue Total RNA Extraction kit (iNtRON) according to the manufacturer's instruction. It (1 ⁇ g) was used in cDNA synthesis reactions performed with Reverse Transcription system (Promega).
  • the amounts of dsyno and ubiquitously expressed Drosophila ribosomal protein gene rp49 mRNAs were measured by PCR (94 0 C 5 min for annealing, 38 cycles of 94 0 C 30sec, 6O 0 C 1 min.
  • dsyno forward 5' - ggtgattggatttgcctactacca - 3' (SEQ ID NO: 7); dsyno reverse: 5' - gatacagggtattcatgttgcgaa - 3' (SEQ ID NO: 8); rp49 forward: 5' - atgaccatccgcccagcatac - 3'; (SEQ ID NO: 9) rp49 reverse: 5' - gagaacgcaggcgaccgttgg - 3' (SEQ ID NO: 10)
  • dsyno was driven in the various tissues via the Gal4-UAS system using the Gal4 drivers containing the various tissue-specific promoters and enhancers, such as gmr-Gal4 ⁇ , Ser-Gal4, MS1096-Gal4, and el6E-Gal4. 1 ) Effect of dsyno overexpression in GE27750:
  • GE 27750 is a line from the GenExel EP library which was generated through random mobilization of an EP element by ⁇ 2-3 transposase (Fig.7). It contains an EP element insertion in the UTR region of CG1937.
  • GE27750 (R) was generated in the process of EP element jumping-out to make a deletion or new insertion mutant of dsyno (Fig.8). In this new insertion EP line, the EP element is inserted 8-10 base pair away from original position and its direction is reversed.
  • GE27750 line is semi-homozygous lethal. (GE27750(R) line is homozygous lethal.)
  • Gal4 drivers were crossed with GE27750. These GAL4 drivers express GAL4 protein under the control of various tissue-specific regulatory element and lead to expression of CG1937 in eye, wing or leg. However, no visible phenotype was shown with those Gal4 drivers alone. Thus, it is shown that CGl 937 could be overexpressed with Gal4 driver in this line (Fig.9 and 10). 2) Effect of dsyno overexpression in UAS-dsyno transgenic fly with tissue specific Gal4 Drivers:
  • FRT-GE27750 chromosome For generation of FRT-GE27750 chromosome, GE27750 line was crossed with 82B-W + FRT line and 82B-w + -GE27750 or 82B-w ' -GE27750 chromosome were generated by meiotic crossing-over (CO) between homologous chromosomes in Fl progenies. CO chromosomes were recovered in the F2 generation. The presence of FRT in CO progenies was tested by ability to grow in G418 media and confirmed by PCR using FRT specific primers. The presence of GE27750 was identified by its homozygous lethality. Three 82FRT- W + - GE27750 CO lines and four 82FRT -w " - GE27750 CO lines were recovered.
  • Dmp53 Dorosophila homolog of p53
  • SEQ ID NO: 11 A DNA sequence encoding Dmp53 is shown in SEQ ID NO: 11.
  • An amino acid sequence of Dmp53 is shown in SEQ ID NO: 12.
  • a transgenic fly containing UAS-dsyno and UAS-Dmp53 was generated by injection of recombinant DNA at concentration of l ⁇ g/ ⁇ L into Drosophila melanogaster embryos according to standard procedures (Spradling, 1986).
  • the expression of dsyno and Dmp53 were driven in the posterior half of the wing via the Gal4-UAS system using the el6E-Gal4 drivers containing the tissue-specific promoters and enhancers.
  • the el6E-Gal4/UAS-Dmp53; UAS-dsyno/+ fly was generated from the cross between el6E-Gal4/UAS-Dmp53 females and el6E-Gal4/CyO; UAS-dsyno/TM2 males.
  • Dmp53 expression in the posterior half of the wing blade showed the bubbled wing phenotype (Fig. 27) caused by uneven cell growth between anterior and posterior half of the wing.
  • Wing disc were dissected in PBS, fixed in a buffer of 50 mM Tris-CHl (pH6.8), 1 mM EGTA, 1% Triton X-IOO 5 2 mM MgSO 4 , 150 mM NaCl, 2.2% formaldehyde for 15 minutes, and blocked using a blocking buffer of 50 mM Tris-HCl (pH6.8), 150 mM NaCl, 0.5% NP40 and 5 mg/mL BSA. The fixed wing disk was incubated overnight at 4°C in a 1 :200 dilution of rabbit anti-Dmp53 antibody.
  • the transgenic fly of the present invention find use in a variety application, including: as tools for use in the elucidation of mechanism of particular diseases such as RA and cancer; as a screening tool that identifies therapeutic compounds for use in the treatment of particular diseases such as RA and cancer.
  • the transgenic fly of the present invention find particular use in screening methods designed to identify therapeutic agents for use in the treatment of particular diseases such as RA and cancer.
  • the etiology of RA including overgrowth of RSCs via overexpressed Syno violin would be proved by the fly as the model organism.
  • Kaneko, M., Tomita, T., Nakase, T, Ohsawa, Y, Seki, H. 5 Takeuchi, E., Takano, H.,
  • Drosophila p53 is a structural and functional homolog of the tumor suppressor p53.

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1352961A1 (en) * 2000-12-22 2003-10-15 Locomogene, Inc. Synovial cell protein
WO2004039999A2 (en) * 2002-10-30 2004-05-13 Syngenta Participations Ag Essential dna enclosed proteins of drosphophilia melanogaster
WO2005061007A1 (ja) * 2003-12-24 2005-07-07 St. Marianna University School Of Medicine 癌の抑制方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1352961A1 (en) * 2000-12-22 2003-10-15 Locomogene, Inc. Synovial cell protein
WO2004039999A2 (en) * 2002-10-30 2004-05-13 Syngenta Participations Ag Essential dna enclosed proteins of drosphophilia melanogaster
WO2005061007A1 (ja) * 2003-12-24 2005-07-07 St. Marianna University School Of Medicine 癌の抑制方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AMANO TETSUYA ET AL: "Synoviolin/Hrd1, an E3 ubiquitin ligase, as a novel pathogenic factor for arthropathy", GENES AND DEVELOPMENT, COLD SPRING HARBOR LABORATORY PRESS, PLAINVIEW, NY, US, vol. 17, no. 19, 1 October 2003 (2003-10-01), pages 2436 - 2449, XP002275253, ISSN: 0890-9369 *
FOLBERG-BLUM ADRIANA ET AL: "Overexpression of mouse Mdm2 induces developmental phenotypes in Drosophila", ONCOGENE, vol. 21, no. 15, 4 April 2002 (2002-04-04), pages 2413 - 2417, XP002357216, ISSN: 0950-9232 *

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