JPH0767500A - Transgenic animal with neurotrophin-3 gene - Google Patents

Abstract

PURPOSE:To obtain a transgenic animal which is given by transferring an exogenous neurotrophin-3 gene, thus useful as an experimental animal model for neuropathies and for research on the neuropathy onset mechanism, development of medisines, and a model animal of hypermegakaryocytosis. CONSTITUTION:The objective transgenic animal having an exogenous neurotrophin-3 (NT-3) gene. In order to transfer NT-3 gene to the target animal, for example, mouse NT-3 gene which has high similarity to human NT-3 gene is connected to the downstream of the promotor which can be expressed in mammalian cells to constitute a gene contract. The gene contract is micro- injected into the fertilized ovum of the target animal. The target animal is preferably a mouse. The promotor is the enolase gene promotor which is specifically expressed in brain.

Description

Detailed Description of the Invention

[0001]

The present invention relates to gene transfer
Genic) regarding animals.

[0002]

BACKGROUND OF THE INVENTION Transgenic animals have a gene introduced into the germ line of the animal or the ancestor of this animal at an early (usually single cell) developmental stage. Wagner (Wag
(1981, Proceedings of National Academy of Sciences (PNA).
S. U. S. A. ) Volume 78, page 5016) and Stewart et al. (1982, Science, Volume 217, page 1046)
Transgenic mice containing the human globin gene are described. Constantine
i) et al. (1981, Nature) 29th
Vol. 4, p. 92) and Lacy et al. (198).
3, Cell, Vol. 34, p. 343) describes transgenic mice containing the rabbit globin gene. McKnight, etc. (198
3, Cell 34, 335) describe transgenic mice containing the transferrin gene. Brinstar et al. (1983, Nature, 306, 332) describe transgenic mice that contain functionally translocated immunoglobulin genes. Palmiter (19)
82, Nature 300, p. 611) describe a transgenic mouse containing the rat growth hormone gene fused to a heavy metal-inducible metallothionein promoter sequence. Palmiter et al. (1982, Cell No. 29)
Vol., P. 701), describes transgenic mice containing a thymidine kinase gene fused to a metallothionein promoter sequence. Palmiter, etc. (1983,
Science 222, p. 809) describe gene transfer mice containing the human growth hormone gene fused to a metallothionein promoter sequence. on the other hand,
Endogenous growth factors that maintain the survival and function of nerve cells and extend according to this concentration gradient are known, and are called neurotrophic factors. Several factors are known as this factor, but nerve growth factor (NGF)
And neurotrophin-3 (JP-A-3-20489).
No. 7) belongs to this factor. Neurotrophin-
Three genes are known to be genes that maintain neurite induction and nerve cell survival in nerve cells,
For example, amendments (1990, Feve's Letter (FEB
Sletter), 266, 187), reports that neurotrophin-3 has a survival-maintaining action and a neurite-stretching action on dorsal root ganglion cells.

[0003]

[Problems to be Solved by the Invention] Neurotrophin-
Elucidation of the physiological function of 3 (hereinafter sometimes abbreviated as NT-3) is considered to be useful for exploring the onset mechanism of nervous system diseases such as dementia or for developing therapeutic agents therefor. The present invention is intended to provide a non-human mammal having an exogenous NT-3 gene transferred, as an animal model for such research and development.

[0004]

The present invention has been completed to solve the above problems. That is, the present invention provides (1) a non-human mammal having an exogenous neurotrophin-3 gene, (2) an exogenous nerve growth factor (hereinafter,
(1), which has a promoter of NGF) and an exogenous neurotrophin-3 gene.
The animal described in (3) above, wherein the non-human mammal is a rodent, the animal described in (1) above, (4) the animal described in (3) above in which the non-human mammal is a mouse, (5) exogenous neuron. The animal according to (1) above, wherein the trophin-3 gene is a human neurotrophin-3 gene, and (6) the animal according to (2) above, wherein the exogenous nerve growth factor promoter is a mouse nerve growth factor promoter. .

The transgenic animal of the present invention has a germ cell and a somatic cell in a non-human mammal or an ancestor of this animal at the germ stage (preferably at the stage of a single cell or a fertilized egg cell and generally at a stage of about 8 cells). Previously) by transferring the NT-3 gene. The NT-3 gene is a gene encoding NT-3 having an action of prolonging the survival of nerve cells or an action of inducing the extension of neurites, which gene is described in JP-A-3-204897. There is. Examples of non-human mammals that can be used in the present invention include cattle, pigs, sheep, goats, rabbits, dogs, cats, and rodents (eg, mice, rats). Of these, rodents, especially mice, are the most preferred target animals.

The exogenous NT-3 gene of the present invention is
It may be derived from mammals of the same or different species from the target animal. In transferring the NT-3 gene into a target animal, it is generally advantageous to use the gene as a gene construct linked downstream of a promoter that can be expressed in animal cells. For example, when the human NT-3 gene is transferred, the NT-3 gene derived from various mammals (porcine, rabbit, rat, mouse, etc.) having high homology with the human NT-3 gene was linked to the downstream of various promoters capable of expressing in animal cells. By microinjecting the gene construct into a fertilized egg of a target mammal, for example, a mouse fertilized egg, a transgenic mammal that highly produces NT-3 can be produced. As this promoter, for example, a virus-derived promoter or a ubiquitous expression promoter such as metallothionein can be used, but preferably an NGF gene promoter or an enolase gene promoter which is specifically expressed in the brain is used.

The foreign NT-3 gene is prepared by a method known per se. For example, as the human NT-3 gene, the plasmid pTB1388 described in JP-A-5-103675 (see FIG. 1) is used as a starting material. Can be used as The gene construct with the promoter can be prepared by a usual genetic engineering technique. Transfer of the NT-3 gene at the fertilized egg cell stage is ensured to be present in all germinal and somatic cells of the target mammal. The presence of the NT-3 gene in the germ cells of the created animal after gene transfer means that all the progeny of the created animal have the NT-3 gene in all of their germ cells and somatic cells. The offspring of this type of animal that inherited the gene had NT-3 in all of its germ cells and somatic cells.
Have a gene.

The exogenous NT-3 gene-transferred mammal of the present invention can be subcultured in a normal breeding environment as the gene-bearing animal after confirming that the gene is stably retained by mating. Further, by mating male and female animals carrying the target gene, homozygous animals having the transgene on both homologous chromosomes are obtained, and by mating these male and female animals, all offspring have the extra gene. So that they can be passaged for breeding. NT of the present invention
In a non-human mammal to which the -3 gene has been transferred, NT-3 is highly expressed. Excessive movement of nerve cells causes exhaustion of nerve cells, resulting in degeneration and loss of nerve cells, etc. It may become a disease and can be used as a model animal for its pathological condition. For example, the mouse of the present invention can be used to evaluate a candidate drug for treating a neurological disease. Also, as another possibility,
It is considered that the mouse highly expressing NT-3 gene serves as a model for learning and memory enhancement. In that case, the usefulness of NT-3 will be clarified, and it is expected that the inducer from NT-3 will be used as a method for determining whether or not it is effective as an anti-dementia drug. Further, for example, it is also possible to confirm the action of NT-3 on nerve cells by subjecting this mouse to treatment such as cerebral ischemia according to a conventional method and examining whether or not nerve cell death due to this treatment can be suppressed. is there.

The transgenic mammals of the invention can be used as a cell source for tissue culture. For example, the neurotrophic factor NT-3 can be analyzed by directly analyzing DNA or RNA in the tissue of the transgenic mouse of the present invention or by analyzing the protein tissue expressed by the gene. N
Cells of tissues containing the T-3 gene can be cultured by standard tissue culture techniques and used to study the function of cells from generally difficult to culture tissues, such as those derived from brain or peripheral tissues. Further, by using the cells, it is possible to select a drug that enhances the function of nerve cells, for example. In addition, if there is a highly expressing cell line, NT-3 can be isolated and purified from it. The transgenic mammal of the present invention can be a model animal for megakaryocytosis because NT-3 further has an activity of promoting colony formation of human peripheral blood lymphocytes in a soft agar medium. Further, since NT-3 also has a fibroblast proliferation promoting activity, it can be used as a model animal for organ fibrosis. In addition, the present transgenic mammal exhibits nephritis which causes dilation of renal bypass, and thus can be used for drug tests for nephritis.

[0010]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples and Examples. The transformed strain Escherichia coli JM109 / pMNT1 obtained in Example 1 was deposited with the Fermentation Research Institute of Japan under the deposit number IFO 15507.
Deposit number FERM BP-436 from July 6, 993, and also to the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.
3 has been deposited since July 15, 1993. Example 1 Construction of plasmid pMNT1 having human NT-3 gene downstream of control region of mouse chromosomal gene Plasmid pTB1388 containing human NT-3 gene
(Described in JP-A-5-103675) 40 μg of Cl
By digesting with aI and HindIII, separating by agarose gel electrophoresis, and extracting the target DNA from the gel,
About 18 μg of 2.2 kbp fragment was obtained. In addition, about 10 μg was obtained by digesting the plasmid pTB1338 with XhoI and HindIII and extracting the target DNA containing the human NT-3 gene expression unit by the same method as above.
A 1.6 kbp fragment of On the other hand, the control region of the mouse chromosomal gene is Zheng and Heinrich (Molecular Brain Research.
es.) Volume 3, p.133-140, 1988). That is, the probe was prepared by the following procedure. 0.5 μl of DNA oligomer

[Chemical 1] And 1 μl H ₂ O, 8 μl buffer (0.625M
Tris-HCl (pH 7.5), 1.25M NaCl, 0.12
5M MgCl ₂ , 0.125M DTT) and mixed
It was treated at ℃ for 15 minutes. After left at room temperature for 15 minutes, the mixture was reacted at 14 ° C. for 1 hour to anneal the oligomer.
After annealing 1 μl of 100 mM dGTP, 1 μl
100 mM dTTP, 24 μl of H ₂ O was added to make the total volume 36 μl. Take 12 μl from this, 10
μl of ³² P-dCTP (~ 3000 Ci / mmole), 1
0 μl of ³² P-dATP (~ 3000 Ci / mmole),
37 with 2 μl of DNA polymerase Klenow fragment
The reaction was performed at 1 ° C for 1 hour. The labeled product was prepared using a nick column (Pharmacia). Using this probe, mouse genome library (Clontech)
From Molecular Cloning (Molecula
r Cloning) (published by Cold Spring Harbor Co.) to obtain a phage containing a control region of mouse NGF chromosomal gene. 100 ng of 3.5 kbp BamHI fragment containing the obtained control region
It was treated with NA polymerase Klenow fragment to create blunt ends. 20 ng of this fragment was added to pBluescript
IIKS (-) (Toyobo) was fragmented with HincII 1
ng was ligated with a Takara ligation kit (manufactured by Takara Shuzo), and E. coli JM109 (manufactured by Nippon Gene) was transformed with this reaction solution to obtain an ampicillin-resistant strain. One of the plasmids possessed by this resistant strain is Mole
color Cloning (Cold Spring
Prepare 100 μg by the method described in
The DNA was cleaved with ClaI and XhoI to obtain about 50 μg of the target DNA. 2.2 kb mentioned earlier
20 ng of 1.6 ClaI-HindIII fragment.
80 ng of the Kbp XhoI-HindIII fragment and 30 ng of the 3.5 kbp ClaI-XhoI fragment were ligated together using a Takara Ligation Kit (Takara Shuzo). Escherichia coli JM109 (manufactured by Nippon Gene) was transformed with this reaction solution to obtain an ampicillin-resistant strain. This transformant (Escherichia coli JM1
09 / pMNT1) plasmid DNA was recovered and digested with restriction enzymes to obtain plasmid pMNT1 (7.3 kb).
p) was obtained. This plasmid contains the 5'upstream region of the mouse NGF gene from about 3.5 kbp to the first exon, and immediately thereafter contains the human NT-3 gene via the 5'untranslated region (170 bp). The above construction diagram is shown in FIG. The structure was examined by multiple restriction enzyme cleavage and contained no detectable rearrangements.

Example 2 Generation of Transgenic Mouse Containing Mouse NGF-Human NT-3 Fusion The above pMNT1 plasmid was cleaved with ClaI,
Concentration of 1 μg-100 μg / ml, 1-2 μl
Was injected into the male pronucleus of fertilized single-cell mouse eggs while observing under a microscope. Then inject the injected egg with Wagner (W
agner) et al. (1981, Proceedings of
National Academy of Sciences (PN
A. S. U. S. A. ), 78, 5016), and transplanted into oviducts of pseudopregnant reared female mice for implantation. These eggs are C57BL / 6J
x DBA2 F1 mice obtained by mating. A commercially available mouse (CLEA Japan, Inc.) was used as a mouse, and the mouse was allowed to grow until it hatched in a bred female mouse.

Analysis of transgenic mice Each offspring was tested by the polymerase chain reaction method with DNA collected from the tail at 3 weeks of age.
That is, the 23-mer primer 1 in the SV40 intron
(5'-GGTGGGTGCAAATCAAAGAAC-
3 ': SEQ ID NO: 3), and 23-mer primer 2 (5'-CTAGTTTGTG in the SV40 poly A addition site.
The polymerase chain reaction method was performed using GTTTGTCCAAACTC-3 ′: SEQ ID NO: 4).
Furthermore, using the DNA collected from the tail, human NT-3
It was analyzed by Southern hybridization with a ³² P-DNA probe containing the gene sequence (labeled by the random prime method). D from the tail in each case
³² P-labeled EcoRI / S prepared by cleaving NA with BglII and prepared from the human NT-3 gene [FIG. 1].
The caI fragment was tested as a probe. DNA for analysis
Is Hogan et al. (1986, Manipulating.
Approximately 1 cm pieces of tail were extracted by the method described by Mouse Embryo (Cold Spring Harbor). The resulting nucleic acid pellet was washed once in 70% ethanol, dried and resuspended in 200 μl of 10 mM Tris (pH 8.0), 1 mM EDTA.
1 μl of the tail DNA preparation was diluted 50-fold with sterile water and used with Primer 1 and Primer 2 at 94 ° C.
Second, 65 ° C. for 1 minute, 72 ° C. for 1 minute 55 times, and the reaction product was electrophoresed through a 1.5% agarose gel to give 1.
Mice in which a DNA band having a size of 1 kbp was observed were selected. 3 transgenic mice from 41 litters (279-2, 281-4, and 283-2 mice)
was gotten. In addition, 10 μ of these three DNA preparations
The g was completely cut, electrophoresed through a 1.0% agarose gel, and Southern (197).
5, Journal Molecular Biology, 98th
Vol., P. 503) and transferred to a nylon filter. The filter was hybridized to the probe overnight and washed twice with 2xSSC, 0.1% SDS at room temperature and once with 0.1xSSC, 0.1% SDS at 65 ° C. The results of this Southern hybridization method showed that all three generated mice examined retained the infused mNGF-NT-3 fusion. All three produced animals integrated the transgene at three different locations to generate three genetically distinct transgenic mouse strains.

The produced mice were bred with normal mice and the DNA was analyzed for the presence or absence of gene transfer progeny in the three lines obtained. The 279-2 and 283-2 mice did confer gene transfer to their offspring. Especially 283
-2 mice also gave transgenic offspring by multiple crosses. This analysis shows that in 279-2 and 283-2 mice the injected gene was transferred via the germ line. Transcription of genes in brain and kidney was determined by Northern hybridization method. The tissue was washed with 5.0 ml cold saline and total RN.
A to Chirgwin et al. (1979,
Biochemistry, Vol. 18, page 5294) was used to isolate using a modified CsCl gradient. RNA
The pellet was reprecipitated in ethanol and washed once.
Next, poly (A) RNA was added to Aviv et al. (197).
2. Proceedings of National Academy
Oligo-dT- by the method of Of Science (P.N.A.S.U.S.A.) Vol. 69, page 1408).
Poly (A) RNA was concentrated using cellulose and finally recovered by ethanol precipitation. 1 poly (A) RNA
Electrophoresis with 1.5% formaldehyde 1.5% agarose gel, sucking it up with a nylon filter, Thomas (1980, Proceedings of National Academy of Science (PN.
A. S. U. S. A. ) Vol. 77, p. 5201). The previous filter
Hybridized overnight to a ³² P-DNA probe containing the human NT-3 gene sequence, 2xSSC, 0.1% SDS.
Washed twice at room temperature with 0.1xSSC, 0.1% S
It was washed once with DS at 65 ° C. 279-integrated and transferred to the next generation of mNGF-NT-3 fusions
1 and 283-2 mice showed transcription in brain and kidney. This expression level is high in the kidney and 5
More advanced stage of development than 1 week old or 6 weeks old 1
Expression levels were higher in 0- or 16-week-old mice.

The amount of NT-3 in the plasma of transgenic mice carrying the mNGF-NT-3 fusion was analyzed by the sandwich enzyme immunoassay method. 100 μl of blood was collected from the fundus of 12-week-old and 24-week-old mice with a heparinized micropipette. After diluting with 400 μl of physiological saline solution, leave it at 4 ℃ for 2 hours.
After centrifugation at 00 rpm for 2 minutes, the supernatant was used as plasma.
A sandwich enzyme immunoassay method using an anti-NT-3 antibody (3W3) conjugated with horseradish peroxidase was performed on the above sample. NT-3 is mN
It was found only in the plasma of the gene transfer mouse having the GF-NT-3 fusion, and its amount was particularly high in the 279-2 mouse and the 283-2 mouse in which the copy number of the transgene was high [Table 1].

[Table 1] NT-3 content in plasma of NT-3 transgenic and non-transgenic mice ━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━ Transgenic mouse Non-transgenic mouse Mice week NT-3 (pg / ml) ¹⁾ Mice week NT-3 (pg / ml) ━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━ 279-2 24 290 279-1 24 ND 279-2-9 12 310 279-2-11 12 ND 279-2-10 12 350 281- 4 24 15 281-3 24 ND 283-2 24 235 283 24 ND ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ND ; Not detected

Test Example 1 Pathological Search of Brain and Kidney of Gene Transfer Mouse Two gene transfer mice carrying the mNGF-NT-3 fusion were found.
Since it was apparently normal until 8 weeks of age, it was examined in more detail. Make thin slices of mouse brain and kidney,
The cells expressing the human NT-3 gene were analyzed by the in situ hybridization method as shown in Takami et al. (1992, Experimental Brain Research, Vol. 90, page 1). For the probe, a 45-mer synthetic DNA having a nucleotide sequence corresponding to the antisense of the human NT-3 gene from nucleotide number 263 to nucleotide number 307 was labeled with α- ³⁵ S-dATP by terminal deoxynucleotidyl transferase. Was used.
The slices were sliced to a thickness of 20 μm and stored on a siliconized glass slide at −80 ° C. until use.
Allow slide glass to stand at room temperature for 10 minutes before use.
It was washed with a phosphate buffer (PBS) containing% formaldehyde for 30 minutes, and further washed with PBS. After treatment with acetic anhydride containing 1.5% triethanolamine for 10 minutes,
It was dried with an ethanol series and treated with chloroform for 5 minutes. Hybridization was performed overnight with the labeled probe in a solution containing 50% formaldehyde. After treatment with a 4 × SSC solution at room temperature for 3 minutes and a 1 × SSC solution at 55 ° C. for 60 minutes, it was dried with an ethanol series and exposed with an X-ray film or emulsion.

In 28-week-old 279-2 and 283-2 mice, the human NT-3 gene was expressed in a small number of neurons and a small number of glial cells in the CA3 region of the hippocampus of the brain by the in situ hybridization method. Admitted. In contrast, its expression was not observed in littermate control mice that were not transgenic mice. When in situ hybridization was performed on kidney sections of the same mouse, its expression was observed in epithelial cells in the bent portion of the distal tubule only in the transgenic mouse. In addition, dilation of the renal bypass was observed in the transgenic mice. This expansion of the renal detour correlated with the copy number of the transgene fusion. This rat was found to be a murine exhibiting renal inflammatory conditions resulting in dilation of the renal bypass.
Drugs expected to have an effect on nephritis can be tested as follows. When the renal inflammatory condition is mild, it is possible to select a gene-transferred mouse most likely to exhibit the renal inflammatory condition and test the drug. The selected animals and their progeny are used as test animals, where they are exposed to a drug which is expected to have an effect on nephritis and the inhibition of dilatation of the renal bypass is measured as an indication of a reduction in renal inflammation. The type and concentration of drug used for selection can be changed to select an appropriate administration concentration.

Test Example 2 Learning Impairment Test of Transgenic Mice NT-3 is a gene highly expressed in the hippocampus of the brain, and its association with higher brain functions such as learning memory has attracted attention. Learning ability was analyzed by performing passive avoidance learning. In the test, male transgenic mice containing the mNGF-NT-3 gene fusion of 7 to 15 months of age and littermate control mice were used. Step-
Through type passive avoidance learning device (dark room; 25x2
5 × 30 cm, bright room; 9 × 9 × 25 cm, guillotine door; 5 × 5 cm). In the acquisition trial on the first day, the acclimation trial to the device was performed 1 to 2 hours before the acquisition trial. The mouse was placed in a light room, the guillotine door was opened 20 seconds later, the four limbs of the mouse were completely moved to the dark room, and then left in the dark room for 10 seconds to be taken out. The acquisition trial was performed by the same procedure, but an electric shock (1 mA) was applied from the floor grid for 3 seconds immediately after the mouse was moved to the dark room. The retention test was performed 24 hours after the acquisition trial, and the latency until the mouse moved to the dark room was measured up to 300 seconds. The average response latencies of acquisition trials and retention tests in passive avoidance learning are shown in [Fig. 4]. Although there was no effect of aging on the response latency of the acquisition trial, the difference in the group of transgenic mice was longer than that in the control group at any age. In retention tests, most mice in both groups showed avoidance of 300 seconds at 7 to 11 months of age, but 12 to 1
At the age of 5 months, the average avoidance latency tended to be shortened in the control group. The above results indicate that the control mice show a tendency for the learning ability to decrease with aging, whereas the mNGF-NT-3 gene-transferred mice have this tendency suppressed. From the above results, it is considered that the mice highly expressing the NT-3 gene may have enhanced learning and memory ability, and the usefulness of NT-3 may be clarified. Therefore, it is expected that NT-3 or its inducer will be effective as an anti-dementia drug.

Test Example 3 Measurement of NT-3 production amount Sandwich enzyme was used to determine the amount of NT-3 in urine, plasma, hippocampus and kidney of the gene transfer mouse (283-2 mouse) obtained in Example 2 at each developmental stage. The results of analysis by the immunoassay method are shown in FIGS. Urine was centrifuged at 12000 rpm for 5 minutes to remove precipitates and used as a sample. For plasma, 100 μl of blood was collected from the fundus of a mouse using a heparinized micropipette, and 300
PB containing μl of 2 mg / ml EDTA, 0.5% BSA
After diluting with the S solution, centrifugation was performed at 4000 rpm for 2 minutes, and the supernatant was used as a sample. 50 mM for hippocampus and kidney
Tris-HCl buffer (pH 7.5), 0.1% Triton (Trito
n) X-100, 0.01% thimerosal, 2% BSA, 4
After extraction with an extraction solution containing 00 mM NaCl and 2 mM EDTA, centrifugation was performed at 12000 rpm for 2 minutes, and the supernatant was used as a sample. A sandwich enzyme immunoassay using an anti-NT-3 antibody (3W3) conjugated with horseradish peroxidase was performed on the above sample. In transgenic mice, urinary NT-3 was detected at 5 weeks of age, and at 20 weeks of age, a maximum of 25.0 ng / ml was measured, which tended to increase with the development of the mouse.
(Fig. 3), in the normal mouse as a control, the amount of NT-3 in urine was below the detection limit. NT- in plasma
3 was detected from the age of 10 weeks in the transgenic mouse, and its amount showed an increasing tendency with the development of the mouse, and a maximum of 2.0 ng / ml was measured at the age of 22 weeks (Fig. 4), but in the control mouse. The amount of NT-3 was below the detection limit. The amount of NT-3 in the transgenic mouse in the hippocampus was always higher than that in the control mouse, and the difference was remarkable at 10 weeks of age. The amount of NT-3 in the kidney was below the detection limit in the control mouse, whereas it was high in the transgenic mouse, and the transition of the amount of NT-3 in the developmental stage was similar to that of urine. It was

[0019]

INDUSTRIAL APPLICABILITY The non-human mammal to which the exogenous NT-3 gene of the present invention has been transferred is used as a pathological model of neurological disease.
It can be used for screening of the therapeutic agent, supply of cells highly expressing NT-3, and model animal for megakaryocytosis.

[0020]

[Sequence list]

Sequence number (SEQ ID NO): 1 Sequence: Sequence length (SEQUENCE LENGTH): 36 Sequence type (SEQUENCE TYPE): Nucleic acid (nucleic acid) Number of strands (STRANDEDNESS): Single strand (single) Topology- ( TOPOLOGY): Linear sequence type (MOLECULE TYPE): other nucleic acid
acid) Antisense (ANTI-SENCE): Yes Sequence: GAGCCGCTGG AGCCGGAGGGGGAGCCGCATCG AGT
GAC 36

Sequence ID (SEQ ID NO): 2 Sequence: Sequence length (SEQUENCE LENGTH): 32 Sequence type (SEQUENCE TYPE): Nucleic acid Number of strands (STRANDEDNESS): Single strand (single) Topology- (TOPOLOGY): linear sequence type (MOLECULE TYPE): other nucleic acid
acid) Antisense (ANTI-SENCE): Yes Sequence: TCCAAATATA AGGCCAGCTA CAAAGTCACC GA 32

Sequence ID (SEQ ID NO): 3 Sequence: Sequence length (SEQUENCE LENGTH): 20 Sequence type (SEQUENCE TYPE): Nucleic acid Number of strands (STRANDEDNESS): Single strand (single) Topology- (TOPOLOGY): linear sequence type (MOLECULE TYPE): other nucleic acid
acid) Antisense (ANTI-SENCE): Yes Sequence: GGTGGTGCAA ATCAAAGAAC 20

Sequence number (SEQ ID NO): 4 Sequence: Sequence length (SEQUENCE LENGTH): 23 Sequence type (SEQUENCE TYPE): Nucleic acid Number of strands (STRANDEDNESS): Single strand (single) Topology- (TOPOLOGY): linear sequence type (MOLECULE TYPE): other nucleic acid
c acid) Antisense (ANTI-SENCE): Yes Sequence: CTAGTTGTGG TTTGTCCAAA CTC 23

[Brief description of drawings]

FIG. 1 shows a construction diagram of a plasmid pMNT1 in Example 1.

FIG. 2 shows the average response avoidance time of acquisition trials and retention tests in passive avoidance learning.

FIG. 3 shows changes in the amount of urinary NT-3 in the developmental stage of NT-3 transferred mice.

FIG. 4 shows changes in plasma NT-3 levels during development of NT-3 transferred mice.

FIG. 5 shows changes in the amount of NT-3 in hippocampal tissues at the developmental stage of NT-3 transferred mice.

FIG. 6 shows changes in the amount of NT-3 in renal tissues at the developmental stage of NT-3 transferred mice.

[Explanation of symbols]

In FIG. 1, in the plasmid pTB1388, the dotted line indicates the SV40 intron and the SV40 poly (A) addition site. The blackened portion shows the human NT-3 gene portion. The solid line indicates the origin of replication of E. coli and the drug resistance gene. ⇒ indicates the transfer direction. In FIG. 2, the white part shows the average reaction recovery time of the acquisition trial, and the shaded part shows the average reaction time of the retention test. 5 and 6, black bars are N.
White bars in T-3 transferred mice represent the amount of NT-3 in control mice. In FIG. 5, NT indicates untested.

Claims (6)

[Claims] 1. A non-human mammal having an exogenous neurotrophin-3 gene. 2. An exogenous nerve growth factor promoter and an exogenous neurotrophin-3 gene.
The listed animals. 3. The animal according to claim 1, wherein the non-human mammal is a rodent. 4. The animal according to claim 3, wherein the non-human mammal is a mouse. 5. The animal according to claim 1, wherein the exogenous neurotrophin-3 gene is a human neurotrophin-3 gene. 6. The animal according to claim 2, wherein the exogenous nerve growth factor promoter is a mouse nerve growth factor promoter.