JP2001103869A - Lipocalin-type prostaglandin d synthetase transgenic animal and examination using the animal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transgenic animal holding a transduced human L-PGDS gene and having congenital or reactive disorder in the central nervous system function, circulatory system function, reproduction function, etc., by the expression of a large amount of the synthetic enzyme. SOLUTION: The transgenic animal is a non-human animal obtained by the ontogenesis of a totipotency cell of a non-human animal holding a transduced human L-PGDS gene and its progeny containing the above gene in a somatic cell chromosome and expressing a large amount of human L-PGDS. The test method for various active substances is characterized by the use of the transgenic animal.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The invention of this application relates to a human lipocalin-type prostaglandin D synthase (L-PGD).
S) The present invention relates to a transgenic animal into which a gene has been introduced and various test methods using the animal. More specifically, the invention of this application relates to a transgenic animal exhibiting a congenital or responsive disorder in the central nervous system function, circulatory system function, reproductive function, etc. due to the overexpression of L-PGDS, and the use of this animal. And a method for testing an active ingredient of a preventive or therapeutic agent for the above dysfunction.

[0002]

2. Description of the Related Art L-PGDS (J. Biol. Chem. 260: 12410)
-12415, 1985; J. Biol. Chem. 264: 1041-1045, 1989; Pr.
oc.Natl.Acad.Sci.USA.88: 4020-4024, 1991; Proc.Nat
l.Acad.Sci.USA. 89: 5376-5380,1992; J.Biol.Chem. 26
7: 23202-23208, 1992; J. LipdMediators Cell Signalin
g 12: 257-273, 1995; J. Biol.Chem. 270: 1422-1428, 19
95) is a prostaglandin D ₂ (PGD ₂ : Prostaglandins Leukotrienes E) having various physiological activities.
ssent.Fatty Acids 37: 219-234, 1989; FASEBJ. 5: 257
5-2581, 1991; Adv.Neuroimmunol. 5: 211-216, 1995;
J. Lipd Mediators Cell Signaling 14: 71-82, 1996) and the transport function of vitamin A group which is a cell differentiation factor (Proc. Natl. Acad. Sci. USA. 88: 4020-4024, 1991; J; .
Biol. Chem. 272: 15789-15795, 1997) and is expressed in the central nervous system, circulatory system and reproductive organs (Arch. Biochem. Biophys. 260: 521-531, 1988; Br. J. Op
hthalmology 72: 461-464, 1988; Proc.Natl.Acad.Sci.U
SA. 90: 9070-9074, 1993; Prostaglandins 51: 298, 199
6; J. Neurosci. 16: 6119-6124, 1996), which is secreted into body fluids (Biochem. Biophys. Res. Commun. 203: 1110-1).
116, 1994; Proc. Japan.Acad. 72: 108-111, 1996; Clin
ical Cheminsry 42: 1984-1991, 1996). It is also known that the increase or decrease of the expression level of L-PGDS in each of these organs is closely related to various physical disorders and diseases.

For example, PGD ₂ has the strongest hypnotic action among endogenous sleep substances that have been identified to date, but it is an inhibitor of the synthetic enzyme L-PGDS (Ar
Ch. Biochem. Biophys. 289: 161-166, 1991) results in significant sleep disorders when administered intraventricularly to animals (Proc. Natl.
Acad. Sci. USA 88: 9046-9050, 1991) suggests that insufficiency of L-PGDS synthesis in the brain contributes to insomnia. Also, increasing or decreasing PGD ₂ in the central nervous system results in altered susceptibility to noxious stimuli such as nociceptive stimuli (Brain Res. 510: 26-32, 1990; J. Pharmacol.
Exp. Ther. 278: 1146-1152, 1996; Proc. Natl. Acad.
Sci. USA. 96: 726-730, 1999) and changes in the secretion of progesterone that promotes ovulation induction (Endocrinology 110: 2207-2).
209, 1982).

On the other hand, L-PGDS expression in the circulatory system
For example, L-PGDS may be used in atherosclerotic lesions.
It is known to be specifically expressed (Proc. Natl. Aca
d.Sci.USA. 94: 14689-14694, 1997). This is LP
PGD synthesized by GDS _TwoOf blood coagulation (P
rostaglandins 16: 373-388, 1978)
It is speculated that this is not to close the tube. Follow
Thus, L-PGDS in blood is a marker of arteriosclerosis
Promising. In addition, L-PGDS is used in the near renal cells of the kidney.
It is expressed at a specific site in the duct, but that site is
Is associated with decreased renal function due to excessive salt in
L-PGDS is considered to be a renal
It is thought that it is closely related to the onset mechanism of hypoactivity
There are also.

[0005] Furthermore, it is known that the expression of L-PGDS in the reproductive tract is related to infertility. That is, L-PGDS is produced in the testis and epididymis and secreted into the semen, but the L-PGDS concentration in the semen of a patient with sperm reduction, which is a factor of male infertility, is significantly lower than that in a healthy person ( Biol. Reprod. 58: 600-607, 1998). SP26 reported as a protein related to high fertility in bovine semen is L
-PGDS (Biol. Reprod. 5
8: 826-833, 1998). However, since PGD ₂ is hardly detected in semen (Biol. Reprod. 58: 600-607,
1998), L-PGDS is thought to be related to reproductive function by another function and PGD ₂ synthesis function.

[0006] L-PGD is also involved in female pregnancy. That is, L-PGDS is expressed in the fetal central nervous system in late pregnancy and secreted into amniotic fluid, and it has been confirmed in humans and animals that the amount increases with the growth of the fetus. Therefore, L-PGDS is expected to be used as a marker for abnormal pregnancy.

[0007]

SUMMARY OF THE INVENTION As described above, LP
GDS is closely related to various physiological functions of living organisms, and it has been suggested that its overexpression may cause various human diseases.

[0008] However, as to how the overexpression of L-PGDS activity acts on animal individuals,
No model animal system has been established that allows for research under controlled conditions.

Further, such a model animal is L-PG
It is expected to be extremely effective for the development of drugs for preventing or treating various diseases caused by the overexpression of DS activity.

The invention of this application has been made in view of the above circumstances, and has as its object to provide an animal individual that genetically expresses a large amount of L-PGDS activity. In addition, this application uses this animal individual,
It is another object of the present invention to provide a method for testing the efficacy of a substance for preventing or treating various diseases caused by deficiency of PGDS activity.

[0011]

SUMMARY OF THE INVENTION The present invention is directed to an invention for solving the above-mentioned problems, in which a human lipocalin-type prostaglandin D synthase gene is introduced into a totipotent cell of a non-human animal. The present invention provides a transgenic animal, which is a non-human animal and its progeny, which has the gene in a somatic chromosome and expresses human lipocalin-type prostaglandin D synthase in large amounts.

[0012] In a preferred embodiment of the transgenic animal, the non-human animal is a mouse. Further, this application provides the following various test methods. (1) A method for testing the activity of a sleep regulating substance in an individual, which comprises administering a candidate substance to the transgenic animal and measuring the sleep state of the animal. (2) A method for testing the activity of an analgesic substance in an individual, which comprises administering a candidate substance to the transgenic animal and measuring the responsiveness of the animal to a pain stimulus. (3) A method for testing the activity of an anticoagulant substance in an individual, which comprises administering a candidate substance to the transgenic animal and measuring the degree of arteriosclerosis of the animal. (4) A method for testing the activity of a renal function promoting substance in an individual, comprising administering a candidate substance to the transgenic animal and measuring the renal function of the animal. (5) A method for testing the activity of a fertility-improving substance in an individual, which comprises administering a candidate substance to the male of the above-mentioned transgenic animal, and measuring the degree of maturation or reproductive rate of sperm of the male animal. A test method characterized by the following. (6) A method for testing the activity of an infertility-improving substance in an individual, comprising administering a candidate substance to a female transgenic animal before or after pregnancy, and implanting a fertilized egg in the uterus of the female animal. Alternatively, a test method comprising measuring the state of development of a fetus. (7) A method for testing the activity of an anesthetic substance in an individual, which comprises administering a candidate substance to the transgenic animal, and measuring the anesthesia state and / or arousal state from the anesthesia of the animal. Test method. (8) A method for testing the activity of a physiological disorder improving substance in an individual, which comprises administering a candidate substance to a female of the above-mentioned transgenic animal, and measuring the sexual cycle, ovulation count and / or various hormone levels of the female animal. Test method characterized by performing

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Human L-PGD as a transgene
As the S gene, its cDNA can be used. This L-PGDS cDNA has a known sequence (J. Biol. Chem. 2
67: 23202-23208, 1992), a method for screening a human cDNA library using the oligonucleotide as a probe based on an arbitrary nucleotide sequence, and hybridizing to both ends of a cDNA fragment of interest. And using the primers as primers to RT from mRNA isolated from human cells.
-It can also be prepared by the PCR method.

[0014] A promoter sequence and an enhancer sequence for controlling the expression of the transgene are linked to the transgene. The transgenic animal of the present invention can be prepared by a known transgenic animal production method (for example, Proc. Natl. Aca).
d. Scl. USA 77; 7380-7384, 1980). That is, the transgenic animal of interest is introduced by introducing the transgene into a totipotent cell of a non-human animal, generating the cell into an individual, and selecting an individual having the transgene integrated in the genome of a somatic cell. Can be produced. As a non-human animal, it is technically possible to target all animal species, but in particular many inbred strains have been created, and in addition, techniques such as fertilized egg culture and in vitro fertilization have been established. A mouse that works best. In the case of a mouse, a fertilized egg or an early embryo can be used as the totipotent cell into which the gene is introduced. As a method for introducing a gene into cultured cells, physical injection of DNA (microinjection) is considered in consideration of the yield rate of an individual transgenic animal and the efficiency of transgene transfer to the next generation.
The method is optimal.

The fertilized egg into which the gene has been injected is then transplanted into the fallopian tube of the foster mother, and the animals that have developed and born to the individual are fostered and reared, and then DNA is obtained from a part of the body (tail tip).
And the presence of the transgene is confirmed by Southern analysis or PCR. If the individual in which the presence of the transgene is confirmed is defined as the first generation (Founder), the transgene is transmitted to 50% of its offspring, and a wild-type or mutant animal can be efficiently produced.

Since the transgenic animal thus produced overproduces L-PGDS,
It can be a perfect model to study the physiological effects of D _2. In addition, it can be a model suitable for screening drugs for L-PGDS.

The transgenic animal thus prepared can be used in the following test methods. (1) Test Method for Sleep Regulatory Substance When a L-PGDS activity inhibitor is intracerebroventricularly administered to a wild-type mouse or the like, a remarkable sleep disorder is observed. Presents with transient hypersomnia. This indicates that PGD ₂ contributes to sleep induction under stress and in pathological conditions. Therefore, a candidate substance for a sleep regulating substance to be an active ingredient such as a hypnotic is administered to the transgenic animal,
By measuring the sleep state of animals under stress or pathological conditions, for example, the search for sleep regulating substances including stimulants with few side effects, the development of hypnotics and stimulants containing such substances as active ingredients have been promoted. It becomes possible. In addition, it is possible to elucidate the sleep-wake regulation mechanism in mammals through such a search for a sleep regulating substance. The sleep state of the mouse can be measured, for example, by measuring brain waves, myoelectricity, activity, eating / water consumption, body temperature, and the like over time. (2) Method of Testing Analgesic Substances Humans do not feel pain due to contact stimuli when healthy, but in severe illness such as when suffering from shingles, severe tactile stimuli cause severe pain. This is a phenomenon called allodynia, which is distinguished from hyperalgesia caused by thermal or mechanical stimulation (Textbook of Pain, 3rd
Ed, pp165-200, 1994; Pain 68: 13-23, 1996). Also,
PGD ₂ in the spinal cord are involved in the regulation of expression of allodynia (Proc Natl Acad Sci USA 96: ..... 726-730,
1999).

Therefore, the transgenic animal of the present invention can be used for elucidating the mechanism of inducing allodynia and hyperalgesia. Further, by administering a candidate analgesic substance to a mouse and measuring the responsiveness of the mouse to a pain stimulus, it becomes possible to develop an analgesic which is selective for a pain sensation reaction. Furthermore, elucidation of the mechanism of pain induction and development of novel analgesics are effective in elucidating, for example, the mechanism of severe pain at the end of cancer that cannot be suppressed by morphine, and developing a treatment for the pain. (3) Test method for anticoagulant substances Conventionally, rats and rabbits that forcibly induced arteriosclerosis have been used as arteriosclerosis model animals, but mice have atherosclerosis caused by eating much like humans. (Atheroscleosis 57: 65-73, 1985). Also, as described above, L-PGD is specifically present in atherosclerotic lesions.
It is known that S expresses and lyses coagulated blood by PGD ₂ synthesized thereby to prevent vascular closure. The transgenic animal of the present invention (particularly,
Transgenic mice) express L-PGDS in large amounts, and may be able to reduce attacks of heart failure due to arteriosclerosis.

Therefore, by administering a candidate for a blood coagulation inhibitor to a transgenic animal that has caused arteriosclerosis and measuring the degree of arteriosclerosis in the mouse, the mechanism of arteriosclerosis in humans can be elucidated and a new blood can be obtained. It is possible to develop anticoagulants. (4) Test method for renal function promoting substance As described above, L-PGDS is specifically expressed in a site related to a decrease in renal function. Therefore, this L-PGD
Transgenic animals into which the S gene has been introduced may be less likely to cause renal dysfunction due to, for example, salt intake, and by administering a candidate substance to such a model mouse to measure the renal function of the mouse, It becomes possible to develop new renal function promoters. It is also effective in elucidating the mechanism of onset of renal hypofunction in humans. (5) Test Method for Fertility-Improving Substances Due to Male Factors As described above, the L-PGDS concentration in semen of a spermatozoa patient who is a factor of male infertility is significantly low, but the L level in the semen of a bull showing a high conception rate is high. -PGDS concentration is high. Therefore, by administering a candidate substance to the male transgenic animal of the present invention and measuring the degree of sperm maturation and reproductive rate of the male animal, it is possible to develop a therapeutic agent for infertility. Also,
It is also effective in elucidating infertility due to male factors. (6) Test method for fertility-improving substance due to female factors In wild-type female mice, L-PGDS is specifically and transiently expressed in the placenta in the second trimester. On the other hand, when PGD ₂ is decreased, the delivery is normal, but the gestation period is markedly prolonged. Therefore, by administering a candidate substance before or after pregnancy of the transgenic animal and measuring the implantation state of the fertilized egg in the uterus of the female mouse or the developmental state of the fetus, it is possible to improve infertility due to female factors. Drug development becomes possible. (7) Test method of anesthetic substance In general, general anesthesia is performed using an inhaled anesthetic substance during surgery, etc., but ideal anesthesia is painlessness, muscle relaxation and loss of consciousness due to a low concentration of inhaled anesthetic. Say. However, there are individual differences in the state of anesthesia depending on gender, age, physique, health status, and the like. In patients who are hardly anesthetized, death may be caused by use of a high-concentration inhalation anesthetic. For this reason, the concentration of inhaled anesthetics must be carefully determined.However, since the mechanism of action of inhaled anesthetics on the central nervous system has not been elucidated, medical practice relies on the rules of thumb of experienced physicians. At present it is inevitable. By the way, the decrease in PGD ₂ in the brain is due to the fact that the depth of anesthesia with an inhaled anesthetic substance becomes shallow,
-The mechanism leading to analgesia, muscle relaxation, and sleep can be separately analyzed by measuring the state under anesthesia and / or after arousal in the transgenic animal of the present invention that overexpresses PGDS. . This not only allows more detailed evaluation of the efficacy of currently used inhalational anesthetics, but also allows the development of new inhalational anesthetics. In addition, as an anesthesia state of a mouse, for example, loss of righting reflex, painlessness, muscle relaxation, loss of consciousness, etc. can be measured according to a conventional method. Also,
The arousal state can be measured by, for example, recovery of righting reflex, pain response, recovery of muscle strength, and arousal. (8) Test method of physiological disorder improving substance It is known that ovulation cycle and sleep and activity are synchronized (Gen. Comp. Endocrinol. 7: 10-17, 1996; Physio
l. Behav. 49: 1079-1084, 1991; Brain Res. 734: 275-2
85, 1996; Brain Res. 811: 96-104, 1998). Physiological irregularities occur due to the modulation of life rhythm, but physiological irregularities due to abnormal secretion of hormones cause social life, such as excessive sleep. In addition, animals in which PGD ₂ has been reduced show an estrous cycle in which the period before and after the ovulation period is prolonged. Since PGD ₂ changes the secretion of hormones that promote ovulation induction, the transgenic animal of the present invention is effective for elucidating the ovulation induction mechanism and the induction mechanism such as excessive sleep due to irregular menstruation, and a novel physiological It becomes possible to develop irregular improvement substances.

In each of the test methods described above, transgenic animals having different L-PGDS expression levels (for example, homozygous animals having a transgene in both diploid chromosomes and heterozygous animals having a transgene in one of them) are used. Junction type)
It is possible to analyze in detail the various symptoms depending on the amount of L-PGDS production and the effects of the test substances on them by properly using.

[0021]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. Example 1 (1) Preparation of Transgenic Mouse Human L-PGDS cDNA was cloned from a cDNA library prepared from human cell mRNA using rat L-PGDS gene cDNA as a probe.

Next, the human L-PGDS c was inserted into the cloning site (Sal I / Not I) of the vector (pCAGGS).
The DNA was inserted and ligated to construct a transfer vector. FIG.
Is the structure of the transgene in this transfection vector.

This transfection vector was injected into fertilized eggs of FVB mice by the microinjection method. The transgenic fertilized egg was transplanted into the fallopian tube according to a standard method, developed into an individual, and born.

DNA was extracted from the tail of the obtained mouse individual, and transgenic mice were selected by Southern analysis using a probe synthesized based on the sequence of the transgene. Three independent transgenic mice with different L-PGDS gene expression levels were established. The results are as shown in FIG. (2) Examination of gene expression in transgenic mice The expression of transgenes in whole body of transgenic mice was examined by Southern analysis. As a result, in B20 mice, it was confirmed that the L-PGDS gene was expressed at high levels in skeletal muscle, heart, lung, large intestine, and liver.
The results are as shown in FIG. (3) Examination of PGD enzyme activity in transgenic mice PGD enzyme activities in various organs of transgenic mice were examined using PGH ₂ as a substrate. In various organs, transgenic mice showed a marked increase in enzyme activity. Also, when comparing the three systems,
An increase in enzyme activity was observed in the order of B20>B25> B7.
The results are as shown in FIG. Example 2 Using the transgenic mouse obtained in Example 1, analysis of sleep induction by pain stimulus was performed.

In the transgenic mouse, the stimulus for cutting off the tip of the transgenic mouse resulted in 1 to 1 compared to the wild type mouse.
It was observed that the amount of slow wave sleep after 2 hours was significantly increased. The results are as shown in FIG.

When the brain was excised and the amount of PGD ₂ was quantified, it was found that the concentration increased with an increase in the amount of sleep. The results are as shown in FIG.
From the above results, the transgenic animal of the present invention is useful as a model animal for elucidating the mechanism of sleep induction caused by pathology or stress, and is also effective as a system for screening a novel sleep-wake modulator. confirmed.

[0027]

As described above in detail, according to the present invention, the human L-PGDS gene is transduced, and a large amount of this synthetic enzyme is exerted on the central nervous system function, circulatory system function, reproductive function, etc. Transgenic animals exhibiting sexual or responsive disorders are provided. By using this transgenic animal, it becomes possible to test the active ingredient of the drug for preventing or treating the above-mentioned dysfunction at the animal individual level.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the structure of an introduction vector used for producing the transgenic mouse of the present invention.

FIG. 2 shows the results of Northern blot analysis of mRNAs extracted from various organs of three lines of transgenic and wild-type mice.

FIG. 3 shows the results of Northern blot analysis of mRNA extracted from systemic organs of transgenic mice.

FIG. 4 shows L-proteins using protein fractions extracted from various organs of three lines of transgenic and wild-type mice.
It is an analysis result of PGDS enzyme activity.

FIG. 5 is a graph showing changes in the amount of slow-wave sleep after transection of the tail tip in transgenic mice and wild-type mice. ● is tail cutting, ○ is control.

FIG. 6 is a graph showing changes in the amount of PGD _{2 in} the brain after transection of the tail tip in transgenic mice and wild-type mice.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 C12N 5/00 B 33/88 15/00 A (72) Inventor Yoshihiro Urade Kyoto, Kyoto, Japan 440 Towa City Corp. 706 (72) Inventor Naomi Eguchi 1-8-7, Mizumitsu, Higashiyodogawa-ku, Osaka-shi, Osaka (72) Inventor Hiroaki Kitayama Goryomine, Saikyo-ku, Kyoto-shi, Kyoto 3-5-5 Kadomachi 12-72 (72) Inventor Naoki Hayashi 2838-4 Kamiida-cho, Izumi-ku, Yokohama-shi, Kanagawa F-term (reference) 2G045 AA01 AA08 AA29 CB17 4B024 AA01 AA11 BA07 BA80 CA04 CA09 DA02 EA04 GA12 HA13 HA14 4B065 AA91X AA93Y AB01 AC14 AC16 BA04 BD50 CA44 CA46 CA60

Claims (10)

[Claims] 1. A non-human animal and its progeny obtained by ontogenizing a totipotent cell of a non-human animal into which a human lipocalin-type prostaglandin D synthase gene is introduced, and a progeny animal thereof, wherein Gene
A transgenic animal characterized by expressing a large amount of lipocalin-type prostaglandin D synthase. 2. The transgenic animal according to claim 1, wherein the non-human animal is a mouse. 3. A method for testing the activity of a sleep regulating substance in an individual, the method comprising administering a candidate substance to the transgenic animal of claim 1 or 2, and measuring the sleep state of the animal. Method. 4. A method for testing the activity of an analgesic substance in an individual, which comprises administering a candidate substance to the transgenic animal according to claim 1 or 2 and measuring the responsiveness of the animal to a pain stimulus. Test method. 5. A method for testing the activity of a blood coagulation inhibitor in an individual, the method comprising administering a candidate substance to the transgenic animal of claim 1 or 2, and measuring the degree of arteriosclerosis of the animal. Test method. 6. A method for testing the activity of a renal function promoting substance in an individual, comprising administering a candidate substance to the transgenic animal according to claim 1 or 2 and measuring the renal function of the animal. Test method. 7. A method for testing the activity of an infertility-improving substance in an individual, the method comprising administering a candidate substance to a male transgenic animal according to claim 1 or 2, and subjecting the male animal to sperm maturation and / or A test method characterized by measuring the degree of reproduction rate of the male animal. 8. A method for testing the activity of an infertility-improving substance in an individual, which comprises administering a candidate substance to a female of the transgenic animal according to claim 1 before or after pregnancy, A test method comprising measuring the implantation state of a fertilized egg and / or the development state of a fetus. 9. A method for testing the activity of an anesthetic substance in an individual, comprising administering a candidate substance to the transgenic animal according to claim 1 or 2 and measuring the anesthesia state and / or arousal state from the anesthesia of the mouse. A test method characterized by: 10. A method for testing the in vivo individual activity of a physiological disorder improving substance, which comprises administering a candidate substance to a female transgenic animal according to claim 1 or 2, wherein the female animal has a sexual cycle, ovulation number and / or ovulation rate. Or a test method characterized by measuring the amount of various hormones