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WO2005094571A1 - Animal génétiquement modifié et méthode de mesure de l'exocytose à l'aide de l'animal - Google Patents

Animal génétiquement modifié et méthode de mesure de l'exocytose à l'aide de l'animal Download PDF

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Publication number
WO2005094571A1
WO2005094571A1 PCT/JP2005/006140 JP2005006140W WO2005094571A1 WO 2005094571 A1 WO2005094571 A1 WO 2005094571A1 JP 2005006140 W JP2005006140 W JP 2005006140W WO 2005094571 A1 WO2005094571 A1 WO 2005094571A1
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Prior art keywords
animal
hetero
exocytosis
strain
gene
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Japanese (ja)
Inventor
Hiromu Yawo
Yuchio Yanagawa
Jun-Ichi Miyazaki
Toru Ishizuka
Rikita Araki
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Japan Science and Technology Agency
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Japan Science and Technology Agency
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the present invention relates to a genetically modified hetero animal and a method for measuring exocytosis using the animal.
  • the present invention relates to a method for measuring systematic exocytosis in cells of a living body using a genetically modified animal. More specifically, the present invention relates to a fusion protein of one type of V-SNARE and a fluorescent protein, a hetero animal expressing Cre recombinase, and a method for detecting and quantifying exocytosis using the hetero animal.
  • Substances induced in cells in response to chemical signals of intracellular and external forces are encapsulated in the Golgi apparatus within cells by secretory granule-like membranes (vesicular membranes).
  • the substance contained in the vesicle membrane is released outside the cell through the process of transporting the vesicle membrane to the plasma membrane, adhesion, fusion and opening of the vesicle membrane and the plasma membrane.
  • secretory proteins and the like produced in the endoplasmic reticulum are transported to the cell membrane via the Golgi apparatus and are exocytosed out of the cell.
  • Exocytosis mainly transports high molecular compounds such as proteins to the outside of cells, but sometimes also transports low molecular compounds.
  • synaptic transmission in the central nervous system and muscles occurs through the release of neurotransmitters by exocytosis.
  • vesicles containing neurotransmitters are stored at the end of nerve cells, and the action potential is triggered to cause the vesicles to fuse with the plasma membrane, resulting in neurotransmitters.
  • the released neurotransmitters are the nerve cells receiving synapses. And transmits information to downstream nerve cells.
  • the exocytosed vesicle membrane is recovered by endocytosis and replenished and reused for neurotransmitters.
  • signal transmission is controlled by exocytosis and endocytosis control mechanisms.
  • mast cells that secrete histamine contain many histamine-containing intracellular granules.
  • these intracellular granules fuse with the plasma membrane, and histamine inside the granules is exocytosed. Since the release of histamine in mast cells can be observed under a microscope, it has long been known as an example of observable exocytosis.
  • This SNARE hypothesis is based on the hypothesis of synaptobrevin ZVAMP (synaptobrevin / VAMP).
  • synaptobrevin ZVAMP synaptobrevin / VAMP.
  • 24 SNAREs have been found in yeast and 35 SNAREs in mammals, most of which are C-terminally anchored transmembrane proteins with most of the molecule exposed on the cytoplasmic side. And has a site forming a coiled coil structure called an SNARE motif at a site adjacent to the transmembrane region.
  • VAMP 2 is a representative molecule of V SNARE, and its cytoplasmic (outside the vesicle membrane) structure is a major functional domain of VAMP-2.
  • cytoplasmic (outside the vesicle membrane) structure is a major functional domain of VAMP-2.
  • amino acid residues also exist inside the vesicle membrane, but these amino acid structures are considered to have no function.
  • the pH outside the vesicle membrane is about 7.4 neutral, while the pH of the lumen of the intracellular vesicles is about 5.6 acidic. . This is due to the action of the proton pump present in the membrane of the intracellular vesicle.
  • the green fluorescent protein (GFP) and its variants derived from O jellyfish have been applied to various research purposes as markers for biomolecules, but their fluorescence intensity is known to be pH-dependent. . For example, the fluorescence intensity of EGFP, a typical GFP variant, increases 3.5-fold when the pH changes from 5.6 to 7.4.
  • synaptofluorin a fusion protein of the vesicle lumen domain of VAMP-2, a protein of the synaptic vesicle membrane, and superecliptic pHluorin is forcibly expressed to produce the fusion protein. It can monitor pH changes in the environment surrounding the protein, that is, whether the vesicle membrane is included and released.
  • the vesicles are In the vesicle, that is, when the vesicle membrane is in a subsumed state, almost no fluorescence of synaptofluorin is observed, and when it is fused with the plasma membrane and released (exocytosis), fluorescence is observed, and small fluorescence is observed by endcytosis. It utilizes the principle that fluorescence is no longer observed when the pH of the endoplasmic reticulum returns to its original state and the pH of the endoplasmic reticulum returns to its original state.
  • synabtofluorin has so far been limited to cultured neuronal cells that facilitate gene transfer, and attempts have been made to systematically apply this method to living organs and tissues. Did not.
  • the present invention is to construct a method capable of measuring exocytosis extremely easily. Compared with a conventional exocytosis measurement method, the present invention is much more convenient, specific, and sensitive. Provided is a method for measuring exocytosis which can be repeatedly measured in vivo in a non-invasive manner, and a hetero animal for the method.
  • the present inventors studied the development of a method using experimental animals such as mice in order to elucidate the mechanism of exocytosis and elucidate the dynamics of drugs and bioactive substances in vivo through the mechanism. As a result, they have found that extremely effective experimental model animals can be produced using the conditional targeting method.
  • An expression promoter sequence an arbitrary gene sequence including a poly-A sequence having a ⁇ ⁇ sequence at the 5 'and 3' ends, and a gene sequence encoding a fusion protein of one type of V-SNARE and a fluorescent protein A fusion protein of a V-SNARE and a fluorescent protein obtained by crossing an animal into which a gene obtained by linking in the transcription direction in this order with an animal of the same type capable of expressing Cre recombinase is crossed. Or a heterologous animal capable of expressing the same. [0019] 2) The hetero animal or strain thereof according to 1), wherein the expression promoter sequence is a CAG promoter.
  • the method of the present invention is remarkably superior in convenience, specificity, and detection sensitivity as compared with the conventional exocytosis measurement method.
  • measurement can be performed only by irradiating light, there is an advantage that the measurement is non-invasive and can be repeated in living animals.
  • Cre recombinase gene in a site-specific, time-specific or drug-induced manner, fusion proteins can be site-specifically, time-specifically or drug-induced. Can be expressed. Therefore, the present invention is expected to be continuously used in many research and development fields in the future, and is extremely useful not only in basic research but also in the field of applied research.
  • FIG. 1 schematically shows the principle of the measurement method of the present invention.
  • FIG. 2 schematically shows a method for producing a hetero mouse of the present invention and a structure of a transgene required for the method.
  • FIG. 3 is a photograph, instead of a drawing, showing the expression of a fluorescent protein in the hippocampus region where the slicing power of the brain of the hetero mouse of the present invention was also obtained.
  • FIG. 4 is a photograph, instead of a drawing, showing the expression of fluorescent proteins in the olfactory bulb and accessory olfactory bulb of the hetero mouse of the present invention.
  • FIG. 5 is a photograph instead of a drawing, showing the expression of a fluorescent protein in mast cells obtained by slicing the brain of a hetero mouse of the present invention, and the change in the fluorescence intensity of the fluorescent protein due to various stimuli.
  • Fig. 1 schematically shows a mechanism in which a fusion protein of one type of V-SNARE and a fluorescent protein emits fluorescence by exocytosis in the present invention.
  • the cell membrane is surrounded by double lines.
  • Granular vesicles present in the cells are indicated by round double lines.
  • the oval shape penetrating the membrane portion of the granular vesicle indicates VAMP2, a kind of V SNARE, and the circle before it indicates fluorin (pHluorin), a kind of fluorescent protein.
  • the lower left circle in FIG. 1 indicates that granule vesicles have been formed and started to be transported.
  • FIG. 2 shows an outline of the recombinant gene used for the production of the hetero mouse.
  • This recombinant gene contains the 5'-end downstream of the non-site-specific CAG promoter sequence, which also has the ability to combine the -Avian 13-actin promoter, the CMV-IE enhancer and the Escherichia ⁇ -globin-poly ⁇ addition signal.
  • the nucleotide sequence encoding a certain synaptofluorin is linked (see the upper part of Fig. 2).
  • the nucleotide sequence encoding synaptofluorin used here was reported by Rothman et al., And its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing. This nucleotide sequence is composed of 1113 bp in total.
  • the 13th to 360th portion is a sequence encoding VAMP-2, the 361st to 387th portion is a sequence of peptide linker, and 388 to :
  • the 107th part is the region that encodes a kind of fluorescent protein, superecliptic pHluorin! /.
  • Tg- ⁇ ⁇ ⁇ Transgenic mice incorporating the recombinant gene (hereinafter referred to as Tg- ⁇ ⁇ ⁇ ) were prepared according to the method of Brister et al. (Brister RL, Cell, Vol. 27, pp. 223-1231, 1981). did. Further, a heterozygous mouse was prepared by crossing this Tg-loxP with a transgenic mouse having the Cre recombinase gene under the control of the CAG promoter (X-Cre transgenic mouse in FIG. 2). Fertilized eggs of transgenic mice obtained by crossing Tg- ⁇ with wild-type mice are referred to as FERM P- 19708 on March 2, 2004, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST).
  • AIST Advanced Industrial Science and Technology
  • the heterozygous mouse expressing the synaptofluorin in the present invention is obtained by crossing a mouse generated from the deposited fertilized egg with a transgenic mouse having the Cre recombinase gene. By combining them, they can be manufactured.
  • Cre recombinase is expressed, and the ⁇ sequence recognized by the Cre recombinase is cut off (see the lower part of Fig. 2), whereby synaptofluorin present downstream thereof is expressed. .
  • Fig. 3 a photograph replacing the drawing.
  • the upper left part of Fig. 3 shows the whole sliced brain, the lower part shows an enlarged hippocampus region, and the upper right part shows a further enlarged part.
  • the lower right part of Fig. 3 is an enlarged view of a part. Extremely bright green fluorescence was observed around the hippocampus, and expression in heterozygous mice was confirmed.
  • the olfactory bulb is an organ that is suggested to be useful as a research object for odor information processing
  • the accessory olfactory bulb is an organ that is suggested to be useful as a research object for pheromone sensory information processing.
  • the left part of FIG. 4 is that of the olfactory bulb, the middle part is an enlarged photograph thereof, and the lower part shows the result of immunostaining of the same site.
  • the right side of Fig. 4 shows the accessory olfactory bulb, and the lower part shows the same area immunostained.
  • VAMP-2 is also expressed in mast cells, and is responsible for exocytosis of granules containing allergens such as histamine. Therefore, mast cells collected from the hetero mouse prepared above were observed. The result is shown in FIG. 5 with a photograph replacing the drawing. The left side of the upper part of FIG. 5 shows the collected mast cells, and the right side thereof shows the mast cells observed with a fluorescence microscope. As a result, expression of synaptofluorin was also observed in mast cells. Increased fluorescence intensity was observed with 48Z80, a drug that promotes exocytosis. In addition, an increase in the fluorescence intensity was observed due to the ammodimone which alkalizes the lumen of the histamine-containing granules.
  • the resulting chart is shown in the lower part of FIG.
  • the vertical axis of this chart indicates the fluorescence intensity
  • the horizontal axis indicates time (second).
  • the bar shown to the right shows 100 seconds.
  • Two upward arrows indicate left force of 50 ⁇ g / m 1 shows the time when 48Z80 was added, and the right side shows the time when 50 mmol of ammonium ion was added.
  • VAMP-2 is widely distributed not only in the brain but also in other organs throughout the body, as shown in Table 1 below, and plays an important role.
  • Tissue (cells) Main functions Reference adipocyte Insulin-stimulated glucose transporter Cain et al. 1992; Martin et al. 1996
  • Adipocytes in Table 1 are a type of adipocyte and take up glucose. Therefore, elucidating the endocytosis and exocytosis of these cells is useful for the treatment and prevention of diseases related to hypertrophic diabetes. Insulin stimulation also induces the transport of glucose transporters inside fat cells or skeletal muscle cells to the cell surface, thereby promoting the uptake of glucose into the cells and increasing the glucose concentration in blood. descend. In addition, VAMP-2 controls insulin exocytosis in pancreatic Langerno and beta cells of the islets.
  • the heteroanimals of the present invention can be used not only for basic research on these organ functions, but also for the clarification and development of treatments for diabetes, the elucidation of pathologies such as allergies, and the development of treatments for drugs and the like. Is also useful.
  • the present invention is based on the research and development of technologies to efficiently administer high-molecular-weight drugs that do not pass through the blood-brain barrier into the brain, and technologies to selectively incorporate pile cancer drugs into cancer cells.
  • the use of different types of heterogeneous animals can accelerate this.
  • the animal in the present invention is a non-human animal, preferably a mammal that can be used for non-human genetic modification.
  • animals that can be used in the present invention include rodents such as mice and rats, and egrets and porcupines. Use of mice for which a number of genetic modification techniques have been established is preferred.
  • the strain of the mouse that can be used in the present invention may be any strain that is commercially available without any particular limitation as long as it can be used for gene modification.
  • the mouse into which the Cre recombinase gene under the control of the expression-regulatable promoter used in the present invention has been introduced has the ability to newly produce using a special promoter. It is also possible to use transgenic mice sold.
  • the V SNARE used in the present invention is not particularly limited as long as it is a SNARE protein having a single-vessel vesicle structure, but VAMP-2, which is widely expressed in vivo, is preferable.
  • the fluorescent protein used in the present invention include various fluorescent proteins such as green fluorescent protein and red fluorescent protein.
  • EGFP and superecliptic pHluorin are preferred, since fluorescent proteins having high pH dependency are preferred.
  • a preferred example of the fusion protein of v-SNA RE and one of the fluorescent proteins used in the present invention is a fusion protein of VAMP-2 and superecliptic pHluorin.
  • One synaptofluorin can be mentioned, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO: 1.
  • V-SNARE protein has a structure in which the C-terminal side is inside the vesicle, it is preferable that the fluorescent protein be fused to the C-terminal side.
  • the gene that can be cut off and sandwiched between ⁇ sequences used in the present invention refers to any gene having a ⁇ sequence at each of the 5 ′ end and the 3 end.
  • a genetic engineering technique using a Cre (Cyclization recombination) recombinase derived from a nocteriophage and a ⁇ sequence was reported by Phara et al. In 1993 (Gu Hua, et al., (1993) Cell, 73: 1155-1164).
  • the PI Bataterio phage CreDNA recombinase with a molecular weight of 38 kDa has the function of removing the region between two ⁇ sequences as circular DNA, and joining the nucleic acid sequences outside of both ⁇ sequences across one ⁇ sequence. Then It has also been reported that Cre recombinase promotes the above ligation reaction by recognizing two ⁇ sequences separated by more than 100 kb on one nucleic acid molecule and ⁇ sequences present on different nucleic acid molecules.
  • the conditional targeting method in the present invention means a gene targeting method which is a gene deletion method using the above-mentioned Cre-1 oxP system, and a gene-deficient mouse produced by this method is generally called a conditional knockout mouse. Have been.
  • the arbitrary gene having a ⁇ ⁇ ⁇ ⁇ sequence at each of the 5 'end and the 3' end used in the present invention includes various genes such as CAT and Neo to facilitate the production and selection of transgenic animals.
  • U which prefers to use drug resistance genes.
  • the region sandwiched by ⁇ and the position of the genome into which the gene encoding the fusion protein is introduced there is no particular limitation on the region sandwiched by ⁇ and the position of the genome into which the gene encoding the fusion protein is introduced.
  • the expressed Cre recombinase is at a position where the loxP sequence can be recognized.
  • the method for producing a hetero animal of the present invention is carried out by a conditional targeting method.
  • a ⁇ targeting animal incorporating the synaptofluorin gene was prepared by the transgenic method, and then the mouse was bred with the same animal expressing the Cre recombinase gene to produce the present invention. Hetero animals can be manufactured.
  • a promoter capable of using various expression promoters may be used depending on the fusion protein used in the present invention. It is preferable to select a promoter that facilitates expression of the protein. Also, the final expression of the fusion protein encoded by the gene located downstream of this promoter can be regulated by the activity of a promoter that controls the expression of the Cre gene. It is preferable to use a promoter having a strong expression inducing activity as a promoter for controlling the expression of the gene. Such preferred promoters include the CAG promoter.
  • the promoter upstream of the Cre gene in the present invention may be a CAG promoter, but various promoters that are site-specific, time-specific, or condition-specific may be used. Can be. By using a site-specific, time-specific, or drug-induced promoter as a promoter whose expression can be controlled, synapses can be organ-, tissue-, time-, or drug-induced. Mice expressing tofluolin can be produced.
  • a physical stimulus or a chemical stimulus is given to a heterozygous animal capable of expressing a fusion protein of one type of V-SNARE and a fluorescent protein and a Cre recombinase in the present invention, or a test subject
  • a method of detecting or quantifying exocytosis by intracellular vesicles of the hetero animal by administering the substance and detecting or quantifying the physiological activity of the stimulus or the test substance is useful for screening various drugs.
  • Examples of the physical stimulus include electrical stimulation and injury
  • examples of the chemical stimulus include toxic substances and bioactive substances.
  • a DNA was prepared by ligating a poly A (pA) sequence to the 3, terminal side of the CAT (chloramue-cole acetyltransferase) gene, and further ligating the ⁇ sequence to the 5, 5 and 3 terminal ends.
  • a CAG promoter is ligated to the upstream side of the obtained DNA, and a base sequence encoding synaptoflurion shown in SEQ ID NO: 1 and a polyA (pA) sequence are ligated to the downstream side to prepare Tg- ⁇ .
  • Tg- ⁇ was constructed (see Fig. 2).
  • Transgenic mice having this gene were prepared according to the guidance for preparing transgenic mice (Brister RL, Cell, Vol. 27, pp. 223-231, 1981). The offspring were selected to obtain Tg-loxP.
  • Fertilized eggs of transgenic mice obtained by crossing Tg-loxP with wild-type mice were transformed into FERM P-19708 on March 2, 2004, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. The deposit was transferred to the International Depositary on March 18, 2005, and given the accession number FERM BP-10298.
  • transgenic mouse into which the Cre gene under the control of the CAG promoter has been introduced and the Tg-— ⁇ transgenic mouse are bred, and the mouse expressing the synaptofluorin gene is selected. Hetero mice were obtained.
  • Example 2 The hetero mouse obtained in Example 1 was killed by blood loss after ether anesthesia, and the brain was immediately removed. The brain was washed with a bicarbonate buffered saline (127 mM
  • the hippocampus was removed.
  • the excised hippocampus was ice-cooled in a bicarbonate buffered saline for 1 minute, embedded in agarose gel, fixed on a microslicer, and 200-400 m slices were prepared in the cooled bicarbonate buffered saline.
  • the remaining brain from which the hippocampus was removed was similarly embedded in agarose gel, and slices of the olfactory bulb and accessory olfactory bulb were prepared with a thickness of 200 to 400 ⁇ m.
  • the prepared slices were diluted with bicarbonate buffer saturated with 95% 0, 5% CO gas mixture maintained at 33 degrees.
  • the slice was placed on a slide glass, covered with a cover glass, and observed with a fluorescence microscope (Olympus BX51). Images were digitized with Polaroid DMC 2 using a NIBA fluorescence filter unit (Olympus) and recorded with a computer (Dell Dimension).
  • mice under ether anesthesia were perfused transcardially with ice-cold 0.1 M sodium phosphate buffer (PBS, pH 7.2) containing 4% paraformaldehyde and 0.1% glutaraldehyde. After that, the brain was removed. Brains were fixed in 0.1 M PBS containing 4% paraformaldehyde after an additional hour. Then, the suspension was replaced with 0.1 M PBS containing 30% sucrose, and a suspension section of 50 / zm was prepared using a cryostat, and left at room temperature for 30 minutes in PBS containing 5% normal goat serum. . The sections were then reacted with an anti-EGFP antibody (1: 500) at 4 ° C.
  • PBS sodium phosphate buffer
  • Example 2 Tyrode's solution (134 mM NaCl, 3 mM KC1, 10 mM HEPES-NaOH, 11 mM glucose, ImM CaCl, ImM
  • the cover glass to which the mast cells were attached was placed in a measurement chamber (capacity: 2 ml), and the Tyrode solution was perfused. Fluorescence intensity was measured with a microscope photometer (Olympus OSP-1) using a B excitation filter unit. Timelabs measurement was performed at 1 Hz with an irradiation time of 100 ms and recorded on a combi- ter (NEC PC9801FA).
  • a Tyrode solution containing a mast cell activation factor (substance 48Z80, Sigma-Aldrich) at a concentration of 50 mgZml was perfused for 200 seconds. After washing with a normal Tyrode's solution, the perfusion was stopped, and 80 ml of Shiojiri's ammonium solution (1 M) was added to neutralize intracellular granules.
  • Fig. 5 shows the results.
  • the present invention enables non-invasive, repeated measurement of exocytosis of cells in living animals, and extremely easily measures exocytosis in a site-specific, time-specific or drug-induced manner. It is intended to provide a measurement method that is far superior in convenience, specificity, and detection sensitivity. Therefore, it is possible to analyze the regulation mechanism of exocytosis at the molecular level, and to control the secretion of hormones, metabolism of chemicals such as drugs, transport between proteins and other cells, and basic biological mechanisms such as the transmission mechanism of nerves. It is possible to measure functional functions at the molecular level, it is extremely useful for investigating the causes of various diseases and abnormalities in which these are involved, and for developing methods for coping with them. Is useful.
  • SEQ ID NO: 1 Nucleotide sequence of a gene encoding a fusion protein of VAMP-2 and a fluorescent protein

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Abstract

Il est prévu de fournir une méthode de mesure de l'exocytose, hautement supérieure en termes de commodité, de spécificité et de sensibilité de détection de la méthode d'exocytose existante, qui peut être utilisée dans une méthode de mesure répétée et non invasive in vivo et un animal lié à cela. En fait, une méthode de détection et de quantification de l'exocytose dans les follicules cellulaires en utilisant un animant exprimant une protéine fondue d'un v-SNARE avec une protéine fluorescente et recombinase Cre ou sa souche ; un animal lié à cela ou sa souche à utiliser dans cette méthode ; une méthode de criblage l'utilisant.
PCT/JP2005/006140 2004-03-31 2005-03-30 Animal génétiquement modifié et méthode de mesure de l'exocytose à l'aide de l'animal Ceased WO2005094571A1 (fr)

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WO2018195019A1 (fr) * 2017-04-18 2018-10-25 The Broad Institute Inc. Composition permettant de détecter une sécrétion et procédé d'utilisation
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JP2016014660A (ja) * 2009-04-23 2016-01-28 カリフォルニア インスティチュート オブ テクノロジー 免疫調節物質の同定方法及びシステム
US11419887B2 (en) 2010-04-07 2022-08-23 California Institute Of Technology Vehicle for delivering a compound to a mucous membrane and related compositions, methods and systems
US10772918B2 (en) 2013-05-10 2020-09-15 California Institute Of Technology Probiotic prevention and treatment of colon cancer
US11331335B2 (en) 2015-06-10 2022-05-17 California Institute Of Technology Sepsis treatment and related compositions methods and systems
WO2018195019A1 (fr) * 2017-04-18 2018-10-25 The Broad Institute Inc. Composition permettant de détecter une sécrétion et procédé d'utilisation
JP2021061809A (ja) * 2019-10-17 2021-04-22 公立大学法人横浜市立大学 ｐＨｌｕｏｒｉｎ−Ｓｅｍａ３Ａノックイン非ヒト動物
JP7317296B2 (ja) 2019-10-17 2023-07-31 公立大学法人横浜市立大学 ｐＨｌｕｏｒｉｎ－Ｓｅｍａ３Ａノックイン非ヒト動物

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