WO2005094571A1

WO2005094571A1 - Genetically modified hetero animal and method of measuring exocytosis using the animal

Info

Publication number: WO2005094571A1
Application number: PCT/JP2005/006140
Authority: WO
Inventors: Hiromu Yawo; Yuchio Yanagawa; Jun-Ichi Miyazaki; Toru Ishizuka; Rikita Araki
Original assignee: Japan Science And Technology Agency
Priority date: 2004-03-31
Filing date: 2005-03-30
Publication date: 2005-10-13
Also published as: JPWO2005094571A1

Abstract

It is intended to provide a method of measuring exocytosis which is highly superior in convenience, specificity and detection sensitivity to the existing exocytosis method and can be used in noninvasive and repeated measurement in vivo, and a hetero animal therefor. Namely, a method of detecting and quantifying exocytosis in cellular follicles by using a hetero animal expressing a fused protein of a v-SNARE with a fluorescent protein and Cre recombinase or its strain; a hetero animal or its strain to be used in this method; and a screening method using the same.

Description

Specification

TECHNICAL FIELD 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.

Background art

[0002] In a cell of an organism, a variety of complex chemical reactions such as production and metabolism of many chemical substances are performed. Cells have the function of taking in necessary chemical substances from outside the cell and transporting them to the intracellular organelles, and transporting produced and unnecessary substances to the outside of the cell. The main types of such transport systems include the active transport of membranes called endocytosis and exocytosis, which take up and release substances by incorporation and release by biological membranes. In the context of active transport of cells through the membrane, extracellular transport is called exocytosis, and intracellular transport is called endocytosis, and is a transport system found in all organs and tissues in a living body.

[0003] 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. For example, 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.

[0004] Exocytosis mainly transports high molecular compounds such as proteins to the outside of cells, but sometimes also transports low molecular compounds. For example, synaptic transmission in the central nervous system and muscles occurs through the release of neurotransmitters by exocytosis. In neuronal cells, 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. Is exocytosed. 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. As described above, at the end of nerve cells, signal transmission is controlled by exocytosis and endocytosis control mechanisms.

[0005] In addition, secretion of many hormones is performed by exocytosis of endocrine cells. Insulin, a hormone that regulates glucose metabolism, is synthesized in j8 cells of spleen islets of Langernoens and concentrated in intracellular vesicles. When a secretory stimulus such as an increase in blood glucose level is applied, the vesicle membrane fuses with the plasma membrane, and insulin is exocytosed. It can be said that such blood glucose control is basically performed by exocytosis.

[0006] In addition, antibody secretion from B lymphocytes and histamine secretion from mast cells are also performed by exocytosis. In response to an allergic reaction, mast cells that secrete histamine contain many histamine-containing intracellular granules. In response to secretory stimulation such as allergic antigens, 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.

[0007] Thus, many drugs and environmental factors are thought to control the function of tissues and organs in association with exocytosis, but there is no excellent method that can directly measure exocytosis of cells. Because of the power, it was extremely difficult to study the details of the exocytosis function and the means of controlling it.

[0008] Fusion of the vesicle membrane and plasma membrane in exocytosis is controlled by an extremely complex reaction involving many molecules. The details have not been fully elucidated yet, but the SNARE hypothesis has become the dominant theory for this mechanism today. In 1987, Rothman et al. Discovered NSF (N-ethylmaleimide-sensitive fosion protein) essential for membrane fusion and a cytoplasmic factor SNAP (soluble NSF attachment protein), which works as an adapter with this NSF. We proposed that the membrane protein factor to which is bound is called SNARE (SNAP receptor).

[0009] This SNARE hypothesis is based on the hypothesis of synaptobrevin ZVAMP (synaptobrevin / VAMP). The formation of a complex between the protein group v-SNARE, which has a single transmembrane structure, and the protein group t, such as syntaxin and SNAP-25, present in the plasma membrane, forms the basis of exocytosis. (Sollner T., et al., (1993) Nature, 362: 318-324). To date, 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.

[0010] 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. Several amino acid residues also exist inside the vesicle membrane, but these amino acid structures are considered to have no function.

[0011] Attempts to measure exocytosis by applying the structure of VAMP-2 have been reported (iesenbock G, et al., (1998) Nature «394: 192-195, ban aranarayanan b, et al., ( 2000) Biophysical Journal 79: 2199-2208) ₀ This is a method that uses a fusion protein of VAMP-2 and a fluorescent protein.

[0012] It is known that 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. On the other hand, 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. Further, the Rosuman scan over path of GFP variants (Rothman) et groups have produced Ekuri flop take full Olin _(superec liptic pHluorin) is, pH 5. 6 forces et 7. 20-fold increase in fluorescence intensity against the change of 4 cause.

[0013] Here, 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. In this method, 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. Sankara Naranan (

(Sankaranarayanan) et al. Reported that this method measures exo-endocytosis in real time (Sankaranarayanan S, et al., (2000) Biophysical Journal 79: 2199-2208).

[0014] However, the application of 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.

Disclosure of the invention

Problems to be solved by the invention

[0015] 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.

Means for solving the problem

[0016] 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.

[0017] That is, the present application provides the following inventions.

[0018] 1) An expression promoter sequence, an arbitrary gene sequence including a poly-A sequence having a 末端 οχΡ 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.

[0020] 3) The hetero animal or its strain according to 1), wherein the expression of the Cre gene is controlled by an expression-regulatable promoter.

[0021] 4) The heterologous animal according to 1), wherein expression of the Cre gene is controlled by a CAG promoter.

[0022] 5) The hetero animal or line thereof according to 1), wherein the hetero animal is a mammal.

[0023] 6) The hetero animal or strain thereof according to 5), wherein the mammal is a rodent.

[0024] 7) The hetero animal or strain thereof according to 6), wherein the rodent is a mouse.

[0025] 8) The hetero-animal or the strain thereof according to 1), wherein the hetero-animal is produced by a conditional targeting method.

[0026] 9) The hetero animal or the strain thereof according to 1), wherein one of V-SNAREs is VAMP-2.

[0027] 10) The hetero animal or a strain thereof according to 1), which is a fluorescent protein having a fluorescence intensity of H-dependent.

[0028] 11) The hetero animal or strain thereof according to 10, wherein the fluorescent protein is superecliptic pHluorin.

[0029] 12) A method for detecting or quantifying exocytosis caused by intracellular vesicles, using the hetero animal or the strain thereof according to any of 1) to 11).

[0030] 13) The method according to 12), which is a method for detecting or quantifying exocytosis by organ-specific intracellular vesicles.

[0031] 14) The method according to 13), wherein exocytosis due to intracellular vesicles is detected or quantified when a physical stimulus, a chemical stimulus, or a test substance is given.

The invention's effect

[0032] The method of the present invention is remarkably superior in convenience, specificity, and detection sensitivity as compared with the conventional exocytosis measurement method. In addition, since 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. In addition, by expressing the 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.

Brief Description of Drawings

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.

BEST MODE FOR CARRYING OUT THE INVENTION

[0034] 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. The pH in these granular vesicles is acidic, about 5.6, and the fusion protein emits little fluorescence. And the force that showed the substance being encapsulated and moving in the granule vesicles is the circle above it. Eventually, the granule vesicles reach the cell membrane, fuse, and open. This state is shown by the open circle in the upper part of FIG. At this time, the granule vesicles open and intersect with the outside world, and their pH becomes about 7.4, the same as the outside world. As the pH increases, the fluorescence intensity of the fusion protein increases, where green bright fluorescence can be observed. The granule vesicles (the right side of Fig. 1) that released the substance outside the cell close the opening and move into the cell. By the action of the proton pump, the pH becomes acidic again to about 5.6. As a result, the fluorescence intensity of the fusion protein decreases, and fluorescence cannot be observed again!

Hereinafter, the outline of the production of a hetero animal and the method of using the same in the present invention will be specifically described using a mouse as an example, but the present invention is not limited to these specific examples.

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 β-globin-polyΑaddition signal. A CAT gene with a Ιο (sequence linked to the 3 'end and a sequence consisting of (chloramphene-col' acetyltransferase) and poly A, and further downstream, a fusion protein of one type of V-SNARE and a fluorescent protein 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! /.

[0037] Transgenic mice incorporating the recombinant gene (hereinafter referred to as Tg-χΡο この) 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-ΙοχΡ 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). Deposited at Tsukuba-Higashi 1-chome 1-Chome No. 6), Ibaraki Prefecture, Japan, which was transferred to the International Depositary on March 18, 2005, and given the accession number FERM BP-10298. . 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.

[0038] In this hetero mouse, Cre recombinase is expressed, and the ΙοχΡ sequence recognized by the Cre recombinase is cut off (see the lower part of Fig. 2), whereby synaptofluorin present downstream thereof is expressed. .

Whether or not the fusion protein was expressed in one of the hetero mice was observed with a fluorescence microscope (Olympus BX51). As a result, in the brain, strong expression was found at the mossy fiber terminals of the hippocampus where memory was formed. The result is shown in Fig. 3 as 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.

[0039] Furthermore, the strongest synaptofluorin expression in the brain was observed in the glomeruli of the olfactory bulb and accessory olfactory bulb. The result is shown in FIG. The olfactory bulb is an organ that is suggested to be useful as a research object for odor information processing, and 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.

[0040] 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, and the horizontal axis indicates time (second). As for the time, the bar shown to the right shows 100 seconds. Two upward arrows indicate left force of 50 μg / 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.

[table 1]

Tissue (cells) Main functions Reference adipocyte Insulin-stimulated glucose transporter Cain et al. 1992; Martin et al. 1996

(GLUT-4) vesicle transport from the inside of the cell to the cell surface

adrenal chromaffin cell exocytosis of secretory granules Hohne-Zell et al. 1994

adrenal gland Rossetto et al. 1996

brain Neurotransmitter exocytosis, neuroendocrine Baumert et al. 1989; Elferink et a 1989; Trimble et al. 1990 gastric parietal cell gastric acid secretion Peng et al. 1997

granulocyte (eosinophil) IgE receptor-mediated EGP eosinophil cationic protein) Hoffmann et al. 2001; Feng et al. 2001

Opening release

granulocyte (mast cell) Open release of secretory granules Guo et al. 1998

granulocyte (neutrophil) Open release of some secretory granules Brumell et a 1995; Feng et al. 2001

heart Rossetto et a on 1996

Kidney collecting duct cell Nielsen et al. 1995 on water channel (aquaporin-2) by vasopressin

* Vesicles from inside ffl vesicle to cell surface

kidney glomerular cell Rossetto et al. 1996

liver hepatocyte Rossetto et a on 1996

Lung endothelial cell Endothelial cis through force beolae 'trans rhino Schnitzer et al. 1995

Tosis

pancreatic acinar cell Open release of zymogen granules Braun et al. 1994; Gaisano et al. 1994

pancreatic islet cell insulin secretion Braun et al. 1994; Regazzi et al. 1995; Rossetto et al. 1996 parotid acinar cell amylase secretion Fujita-Yoshigaki et a 1996

skeletal muscle Insulin stimulated glucose transporter Volchuk et al. 1994

Vesicle transport of (GLUT-4) from intracellular to cell surface

smooth muscle Rossetto et al. 1996; Feng et al. 2001

sperm Membrane fusion process during fertilization (acrosome reaction) Ramalho-Santos et al. 2000

thyroid gland Rossetto 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. Therefore, 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.

[0042] In endothelial cells, it is reported that exoendocytosis involving VAMP-2 is responsible for mass transport by transcytosis. Therefore, 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. Examples of 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. In addition, 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.

[0044] 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. Examples of the fluorescent protein used in the present invention include various fluorescent proteins such as green fluorescent protein and red fluorescent protein. In particular, 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.

[0045] In the present invention, the production of a fusion protein of a V-SNARE protein and a fluorescent protein can be performed mutatis mutandis by a conventional method for producing a fusion protein. Since 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.

[0046] The gene that can be cut off and sandwiched between ΙοχΡ sequences used in the present invention refers to any gene having a ΙοχΡ 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 ΙοχΡ 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 ΙοχΡ sequences as circular DNA, and joining the nucleic acid sequences outside of both ΙοχΡ sequences across one ΙοχΡ sequence. Then It has also been reported that Cre recombinase promotes the above ligation reaction by recognizing two ΙοχΡ sequences separated by more than 100 kb on one nucleic acid molecule and ΙοχΡ 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.

[0047] The arbitrary gene having a Ιο 有する 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.

[0048] Further, there is no particular limitation on the region sandwiched by ΙοχΡ and the position of the genome into which the gene encoding the fusion protein is introduced. Preferably, the expressed Cre recombinase is at a position where the loxP sequence can be recognized.

[0049] The method for producing a hetero animal of the present invention is carried out by a conditional targeting method. For example, first, a ΙοχΡ 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.

[0050] In the present invention, as a promoter for controlling the expression of an arbitrary gene sandwiched between the ΙοχΡ sequences, 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.

[0051] 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.

[0052] In addition, 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. Can be applied. Examples of the physical stimulus include electrical stimulation and injury, and examples of the chemical stimulus include toxic substances and bioactive substances. Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1

Production of hetero mouse

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 ΙοχΡ 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-ΙοχΡ. 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.

[0055] The transgenic mouse into which the Cre gene under the control of the CAG promoter has been introduced and the Tg-—οχΡ transgenic mouse are bred, and the mouse expressing the synaptofluorin gene is selected. Hetero mice were obtained.

Example 2

[0056] Synaptofluorin expression in brain slices of transgenic mice

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

twenty two

NaCl, 3 mM KC1, 25 mM NaHCO, ImM HPO, 16 mM glucose, 2 m

3 3 4

(M CaCl, ImM MgCl 2) for 1 minute. Separates the brain into left and right hemispheres,

twenty two

After removal, 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 μm. The prepared slices were diluted with bicarbonate buffer saturated with 95% 0, 5% CO gas mixture maintained at 33 degrees.

twenty two

It was stored for 14 hours in a salt solution.

[0057] 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).

[0058] Iridological observation was performed as follows. Transgenic 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. overnight. After several washes, the sections were reacted for 1 hour with Piotin-dani anti-Egret IgG antibody (1: 200, Vector Laboratories) and reacted with Betastein ABC (Vecstain ABC) kit (Vector Laboratories). Peroxidase was present in 50 mM Tris-monohydrochloride buffer (ρΗ7.5) in 3,3, -diaminobenzidine-tetrahydride mouth chloride (3,3, -diaminobenzidine tetrahydrochloride) (0.01%). It was visualized by reacting with 002% hydrogen peroxide for 15 minutes. Images were recorded with a CCD camera using a Leica microscope (DMRXE DC Viewer) for observation.

[0059] The results in the hippocampus are shown in Fig. 3, and the results in the olfactory bulb and accessory olfactory bulb are shown in Fig. 4.

Example 3

[0060] Mast cell preparation method and measurement of exocytosis

In Example 2, Tyrode's solution (134 mM NaCl, 3 mM KC1, 10 mM HEPES-NaOH, 11 mM glucose, ImM CaCl, ImM

2

MgCl 2) was injected. After massaging the abdomen for 2 minutes, open the abdominal cavity and pour the fluid

2

They were collected with a Xpoit and collected in plastic centrifuge tubes (15 ml). Further, 2 ml of Tyrode's solution was put into the abdominal cavity again, and the same operation was repeated. After centrifugation at 150 X g for 2 minutes at room temperature, the supernatant was removed and the cells were collected. Repeat this three times to further remove cells Washing with a washing solution. Finally, the cells were suspended in about 0.5 ml of Tyrode's solution and stored in ice water. One drop of the cell suspension was left on a cover glass for 30 minutes to allow the mast cells to adhere to the cover glass. This was placed on a slide glass and observed and recorded with a fluorescence microscope.

[0061] 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).

[0062] In order to induce mast cell exocytosis, 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.

Industrial applicability

[0063] 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.

Sequence listing free text

[0064] SEQ ID NO: 1: Nucleotide sequence of a gene encoding a fusion protein of VAMP-2 and a fluorescent protein

Claims

The scope of the claims

[I] An expression promoter sequence, an arbitrary gene sequence including a poly A sequence having a ΙοχΡ sequence at the 5 ′ end and a 3 ′ end, and a gene sequence encoding a fusion protein of one of V SNARE and a fluorescent protein in this order. Hetero-animal capable of expressing a fusion protein of one V SNARE and a fluorescent protein obtained by crossing an animal into which a gene linked in the transcription direction has been introduced and an animal of the same type capable of expressing Cre recombinase Or the system.

[2] The hetero animal or its strain according to claim 1, wherein the expression promoter sequence is a CAG promoter.

[3] The hetero animal or the strain thereof according to claim 1, wherein the expression of the Cre gene is controlled by a promoter whose expression can be regulated.

[4] The expression ability of Cre gene The hetero animal or its strain according to claim 1, which is controlled by a CAG promoter.

[5] The hetero animal or its strain according to claim 1, wherein the hetero animal is a mammal.

[6] The hetero animal or its strain according to claim 5, wherein the mammal is a rodent.

[7] The hetero animal or its strain according to claim 6, wherein the rodent is a mouse.

[8] The hetero animal or the strain thereof according to claim 1, wherein the hetero animal is produced by a conditional targeting method.

[9] The hetero animal or its line according to claim 1, wherein one of V SNAREs is VAMP2.

[10] The hetero animal or its strain according to claim 1, which is a fluorescent protein having a fluorescence intensity of ΔH.

[II] The hetero animal or its strain according to claim 10, wherein the fluorescent protein is superecliptic pHluorin.

[12] A method for detecting or quantifying exocytosis by intracellular vesicles by irradiating the heteroanimal or the strain thereof according to any one of claims 1 to 1 with fluorescence.

13. The method according to claim 12, which is a method for detecting or quantifying exocytosis by organ-specific intracellular vesicles. 14. The method according to claim 12, wherein exocytosis due to intracellular vesicles when a physical stimulus, a chemical stimulus, or a test substance is applied is detected or quantified.