JPH11332417A - Gene-deficient mice and test methods using these mice - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gene in which an L-PGDS gene has been knocked out, and which exhibits a congenital or responsive disorder in the central nervous system function, circulatory system function, reproductive function, etc. due to the deficiency of this synthase. Provide deficient mice. SOLUTION: An early embryo into which a mouse embryonic stem cell in which a lipocalin-type prostaglandin D synthase gene of genomic DNA is homologously recombined with a mutant sequence obtained by replacing exon II with exon V by a neomycin resistance gene is introduced. A descendant of a chimeric mouse generated and produced in an individual in a female mouse, wherein the gene-deficient mouse has a lipocalin-type prostaglandin D synthase gene of germ cell and somatic cell genomic DNA substituted with a mutant sequence And methods for testing various medicinal substances using the gene-deficient mouse.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gene-deficient mouse and various test methods using the gene-deficient mouse. More specifically, the present invention relates to a lipocalin-type prostaglandin D synthase (L-PGDS)
Is knocked out (disrupted of function), and a gene-deficient mouse showing congenital or responsive impairment in central nervous system function, circulatory system function, reproductive function, etc. The present invention relates to a method for testing an active ingredient of a drug for preventing or treating the above-mentioned dysfunction using a gene-deficient mouse.

[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) and changes in the secretion of progesterone that induce ovulation (Endocrinology 110: 22)
07-2209, 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 _TwoBlood coagulation inhibitory effect (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.

[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 the deficiency may cause various human diseases. However, no model animal system has been established that allows studies under controlled conditions on how L-PGDS activity deficiency acts on animal individuals.

[0008] Such a model animal is L-PG
It is expected to be extremely effective also in the development of drugs for preventing or treating various diseases caused by deficiency of DS activity. The invention of this application has been made in view of the circumstances described above, and has as its object to provide a mouse individual genetically deficient in L-PGDS activity.
In addition, this application uses this mouse individual, L-PGDS
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 a lack of activity.

[0009]

SUMMARY OF THE INVENTION The present invention relates to an invention for solving the above-mentioned problems, which is an L-PG of genomic DNA.
A chimera produced by generating an early embryo into which a DS embryo has been introduced into a mouse mouse by introducing a mouse embryonic stem cell homologously recombined with a mutant sequence in which exon II has replaced exon V with a neomycin resistance gene, in a female mouse An offspring of a mouse, which contains L-genomic and somatic cell genomic DNA
Provided is a gene-deficient mouse in which a PGDS gene has been substituted with a mutant sequence.

[0010] The gene-deficient mouse is L-PG
One of the preferred embodiments is that both or one of the DS alleles is replaced with a mutant sequence. Further, this application provides the following various test methods. (1) A method for testing the activity of a sleep regulating substance in an individual, comprising administering a candidate substance to the gene-deficient mouse and measuring the sleep state of the mouse. (2) A method for testing the activity of an analgesic substance in an individual, comprising administering a candidate substance to the above-described gene-deficient mouse and measuring the reactivity of the mouse to pain stimulus. (3) A method for testing the activity of an anticoagulant substance in an individual, comprising administering a candidate substance to the above-described gene-deficient mouse and measuring the degree of arteriosclerosis of the mouse. (4) A method for testing the activity of a renal function promoting substance in an individual, which comprises administering a candidate substance to the gene-deficient mouse,
A test method characterized by measuring the renal function of the mouse. (5) A method for testing the intra-individual activity of a fertility-improving substance, comprising administering a candidate substance to a male of the gene-deficient mouse,
A test method characterized by measuring the degree of maturation or reproductive rate of the spermatozoa of this male mouse to zero. (6) A method for testing the intra-individual activity of a fertility-improving substance, comprising administering a candidate substance to a female of the gene-deficient mouse according to claim 1 before or after pregnancy, and obtaining a fertilized egg in the uterus of the female mouse. A test method comprising measuring an implantation state or a fetal development state. (7) A method for testing the activity of an anesthetic substance in an individual, comprising administering a candidate substance to the gene-deficient mouse according to claim 1, and measuring the anesthesia state and / or arousal state from the anesthesia of the mouse. Characteristic test method. (8) A method for testing the activity of a substance for improving physiological irregularity in an individual, comprising administering a candidate substance to the female of the genetically deficient mouse according to claim 1, and sexual cycle, ovulation number and / or
Or a test method characterized by measuring the amount of various hormones

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The gene-deficient mouse of the present invention
Known target gene recombination methods (gene targeting:
For example, Methods in Enzymology 225: 803-890, 1993)
Can be created as follows, for example. First, exons II to V in the sequence of the isolated L-PGDS gene were replaced with the neomycin resistance gene (Ne
o ^r gene) and replacement, also thymidine kinase gene scan of the herpes virus at the end of the L-PGDS gene (H
SV-tk gene) to create a targeting vector. This targeting vector is introduced into mouse embryonic stem cells (ES cells), and cell genomic DNA
Are selected by homologous recombination of the L-PGDS gene with the mutant sequence in the targeting vector. The selection of such genetically modified cells, random recombination of G418 added to the cell culture medium to remove non-recombinant cells without Neo ^r gene, HSV-tk gene further added ganciclovir remain This can be done by removing the cells. The L-PGDS gene of the selected transgenic cell is a mutant sequence in which the Neo ^r gene has been inserted into its coding sequence, and cannot produce L-PGDS.

Next, the genetically modified ES cells are injected into an early mouse embryo (blastocyst), and the early embryo is developed into an individual in a female mouse to produce a chimeric mouse. Then, the chimeric mouse and the wild-type mouse are bred to produce offspring mice, and among these offspring mice, a mouse individual having a mutant sequence in both or one of the alleles is selected, whereby the present invention Gene-deficient mice can be obtained. Gene-deficient mice having no L-PGDS-producing ability or having a lower L-PGDS-producing amount than wild type can be prepared. As shown in Examples described later, a gene-deficient mouse (homozygote: − / −) in which both of the alleles are replaced with a mutant sequence has a lower L-type compared to a wild-type mouse (+ / +).
-PGDS activity is 10% or less. In addition, a gene-deficient mouse (heterozygote: +/-) in which one of the alleles is replaced with a mutant sequence has about half the amount of L-PGDS produced by a wild-type mouse (+ / +).

[0013] The gene-deficient mouse thus prepared can be used in the following test method. (1) Test Method of 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, but the gene-deficient mouse of the present invention exhibits a sleep disorder as a genetic trait. .
Therefore, by administering a candidate sleep regulating substance as an active ingredient such as a hypnotic drug to a gene-deficient mouse and measuring the sleep state of the mouse, for example, searching for a sleep regulating substance with few side effects, such a substance is effective. The development of hypnotics as ingredients becomes possible. In addition, it becomes possible to elucidate the sleep regulation mechanism in mammals through such a search for a sleep regulator. 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). The gene-deficient mouse of the present invention does not show any allodynia due to artificial manipulation, and exhibits hyperalgesic symptoms to thermal stimulation.

Therefore, the gene-deficient mouse of the present invention
It can be used to elucidate the mechanism of inducing allodynia and hyperalgesia. In addition, 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 that 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 gene-deficient mouse of the present invention comprises L-PGD
Since it produces almost or only about half of S,
Arteriosclerosis symptoms can develop in a more prominent manner.

Therefore, by administering a candidate for a blood coagulation inhibitor to a gene-deficient mouse that has caused arteriosclerosis and measuring the degree of arteriosclerosis in the mouse, it is possible to elucidate the mechanism of atherosclerosis in humans and to obtain a new blood sample. 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
S gene-deficient mice can easily cause renal dysfunction due to, for example, salt intake. By administering a candidate substance to such a model mouse and measuring the renal function of the mouse, a novel renal function can be obtained. It is possible to develop enhancers. It is also effective in elucidating the mechanism of onset of renal hypofunction in humans. (5) Test method for fertility-improving substance due to male factor As described above, the L-PGDS concentration in semen of a patient with sperm hypotension, which is a factor of male infertility, is significantly low. Therefore, the male of the L-PGDS gene-deficient mouse of the present invention is an effective model animal of spermatozoa, and a candidate substance is administered to this mouse, and the degree of sperm maturation and reproductive rate of this male mouse is measured. This enables the development of fertility treatments. It is also effective in elucidating infertility due to male factors. (6) Test method for fertility-improving substance due to female factor In wild-type female mice, L-PGDS is specifically and transiently expressed in the placenta in the second trimester, whereas in the gene-deficient mouse of the present invention, the delivery Is normal but has a markedly longer gestational age. Therefore, this gene-deficient mouse is an excellent animal model of implantation failure of fertilized eggs and fetal growth retardation.A candidate substance is administered before or after pregnancy of the mouse, and the fertilized egg is transferred to the uterus of the female mouse. Measuring bed status or fetal development allows for the development of drugs to improve infertility due to female factors. (7) Test method for anesthetic substance In general, general anesthesia is performed using an inhaled anesthetic substance during surgery, etc., but ideal anesthesia is painlessness, muscle relaxation, and light sleep with a low concentration of inhaled anesthetic. State. 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, but the mechanism of action of inhaled anesthetics on the central nervous system has not been fully elucidated and medical practice has to rely on the rules of thumb of experienced physicians. There is no present. Since the gene-deficient mouse of the present invention has a small depth of anesthesia with an inhaled anesthetic substance,
By measuring the state under anesthesia and / or after awakening, it is possible to separately analyze the mechanisms leading to analgesia, muscle relaxation, and sleep. 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, disappearance of righting reflex, painlessness, muscle relaxation, sleep state, etc. can be measured according to a conventional method. In the awake state, for example, recovery of righting reflex, pain response, recovery of muscle strength, and arousal can be measured. (8) Test method for physiological disorder improving substance It is known that ovulation cycle and sleep and activity are synchronized (Gen. Comp. Endocrinol. 7: 10-17, 1996; Physiol.
Behav. 49: 1079-1084, 1991; Brain Res. 734: 275-285,
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. The gene-deficient mouse of the present invention shows an estrous cycle in which the period before and after the ovulation period is extended,
The estrous cycle is longer than in wild-type mice. Since PGD ₂ is to change the secretion of hormones to encourage ovulation induction, gene-deficient mice of the present invention is effective to elucidate the induction mechanism of hypersomnia due induction feature and menstrual ovulation, novel physiological It becomes possible to develop irregular improvement substances.

In each of the test methods as described above, the homozygous (-/-) and the heterozygous (+/-) of the gene-deficient mouse of the present invention can be appropriately used to determine the L-PGDS production amount. It is possible to analyze in detail the dependent symptoms and the effects of the test substances on them.

[0017]

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 (Generation of Gene-Deficient Mouse) Known rat L-PGDS gene cDNA (J. Biol. Che
264: 1041-1045, 1989), the cDNA of the rat L-PGDS gene was isolated from a cDNA library prepared from mRNA of rat cells. A 15 kb DNA fragment containing the L-PGDS gene (about 3 kb) was cloned from the genomic DNA. Then, a region including exon V from exon II (the active site coding region of L-PGDS) of the L-PGDS gene is Neor ^.
A mutant sequence was prepared by replacing the gene with the HSV-tk gene at about 10 Kb upstream of the L-PGDS gene, and the mutant sequence was inserted into a vector to prepare a targeting vector (see FIG. 1). As shown in FIG. 1, the L-PGDS coding region of the mutant sequence has about 3
kb.

The targeting vector was added to the undifferentiated cultured ES cells (1.2 × 10 ⁷ cells) by electroporation at 48 μl.
The transfected ES cells were obtained at a rate of g / ml.
These cells were plated on a plate, and after 2 days, G418 and ganciclovir were added to the medium and cultured for another 7 days to obtain colonies resistant to G418 and ganciclovir. After individually separating these colonies and further culturing, DNA was extracted and homologous recombination ES cells were selected by Southern blotting.

Next, this homologous recombination ES cell was
The blastocysts of 7BL / 6 mice were injected into blastocysts by a conventional method, transplanted into foster mothers, and developed into individuals. As a result, 10
Chimeric mice were obtained. Among the obtained chimeric mice, male individuals and female wild-type C57BL / 6 mice were crossed to obtain primary (F ₁ ) mice. These F ₁ mice, Southern blot analysis by individual mutated sequence was confirmed in one of diploid chromosomes (♂, ♀) were selected to obtain these mated with second generation (F ₂₎ Mouse .

Finally, from these F ₂ mice, individuals whose mutant sequences were confirmed on both diploid chromosomes (homozygotes) and individuals whose mutant sequences were confirmed on one side (heterozygotes) by Southern blot analysis ) Was selected to prepare a gene-deficient animal of the present invention. In addition, F ₂ mouse of wild-type:
The ratio of heterozygotes: homozygotes is about 1: 2: 1,
The sex ratio was about 1: 1. No fetal lethality was observed in either homozygotes or heterozygotes.

FIG. 2 shows the DN extracted from the tail of each mouse.
A shows the results of Southern blot analysis of wild-type (+ /
+) Was 5 kb, homo-deficient type (-/-) was 3 kb, and hetero-defective type (+/-) was confirmed to express both. FIG.
FIG. 5 shows the results of Northern blot analysis of mRNA extracted from mouse brain, showing that G3PDH (glyceraldehyde-3) was used.
Although no difference was observed in the amount of mRNA of (-phosphate dehydrogenase), the expression of L-PGDS mRNA was observed only in the wild type and the hetero-deficient type.

FIG. 4 shows LP extracted from the brain of each mouse.
It is an analysis result of the enzyme activity of GDS. The L-PGDS activity of the homo-deficient mouse was less than 10% of that of the wild-type mouse, and about 50% of that of the hetero-deficient mouse. Example 2 (Measurement of Pain Sensitivity of Gene-Deficient Mice) The pain sensitivity of the homo-deficient mouse obtained in Example 1 to thermal stimulation under physiological conditions was examined. That is, a PGE ₂ solution (1 to 100 ng / 5 μl), which is a kind of cytokine, is administered into the intrathecal cavity of a mouse, and 30 minutes later, a hot plate 55
C. and the time until the hind limb was released from the plate was measured. As a result, the reaction time was reduced by about 35% as compared with the case where physiological saline was administered as a control, and was not different from the wild type mouse. That is, the gene-deficient mouse of the present invention showed a hyperalgesic response under physiological conditions, but the degree was similar to that of a wild-type mouse.

Next, the hyperalgesic response (allodynia) due to tactile stimulation under pathological conditions was examined. That is,
A PGE ₂ solution (1-100 ng / 5 μl) was injected into the intrathecal cavity of the mouse, and lightly touched the hind limbs and flank with a fine paint brush every 5 minutes from 5 minutes to 50 minutes, avoidance behavior and vocalization of the mouse And other pain reactions were scored and integrated. The results are shown in FIG.
A. In this figure, the score integrated value is assumed to be 100 when the mouse has assumed all pain reactions.
And the degree of allodynia induction was expressed in%. This FIG. 5A
As is clear from the above, the wild-type mouse showed about 80% or more of allodynia, but the gene-deficient mouse of the present invention (homozygous type) did not show any allodynia.

Further, a mixed solution of PEG ₂ and PGD ₂ was intrathecally administered to a gene-deficient mouse, and the effect on allodynia induction was examined. Results are as shown in FIG. 5B, the PGD ₂ near Fen chromatogram concentration in a physiological concentration, PEG ₂ induced Arodeinia is back. However, lower concentrations of PGD ₂ are ineffective and
High concentration again PEG _2-induced allodynia in disappeared.

Further, a gene-deficient mouse in which allodynia was restored by intrathecally administering a PEG ₂ / PGD ₂ mixed solution was injected with 10 times the amount of PGD ₂ of BWA868C (PGD ₂ receptor antagonist) in PEG ₂ / PGD. _{The two} mixed solutions were administered simultaneously. The results are as shown in FIG.
EG _2-induced Arodeinia was significantly reduced. From the above results, the gene-deficient mouse of the present invention is useful as a model animal for elucidating the mechanism of expression of a hyperalgesic response (allodynia) under pathological conditions, and also screens a novel analgesic component. It was confirmed that it was also effective as a system. Example 3 (Measurement of male litter size of deficient mice) Male F ₂ mice obtained in Example 1 (wild-type and homozygous deficient) the same F ₂ mice female (wild-type and homozygous deficient) And the number of offspring was counted.

The results are as shown in FIG. 6. The number of male offspring of homo-deficient mice was significantly (p <0.01) smaller than that of wild-type male mice. From these results, it was confirmed that the male of the gene-deficient mouse of the present invention was useful as an infertility model due to male factors. Example 4 (L during gestation of females of gene-deficient mice
-PGDSmRNA expression level measurement) obtained in Example 1 F ₂ mice female (homo-deficient type, heterozygous deficient and wild-type) is pregnant, from the placenta, amnion and uterus, and from the brain and liver of fetal Extracted LP
GDS mRNA was quantified by reverse transcriptase PCR.

The results are as shown in FIG. In the case of wild-type mice, L-
PGDS mRNA expression was observed. In the fetal brain of a wild-type mouse, m
Although the expression level of RNA increased, mRNA expression was not detected in liver. Furthermore, in the case of wild-type mice, mRNA expression in amniotic fluid was detected from day 14 of gestation, and the amount increased with the growth of the fetus.

On the other hand, in the case of a homo-deficient mouse, L-type was observed in any period and organ examined, including the fetus.
PGDS mRNA expression was not detected. Example 5 (Measurement of gestation deficient mice) male and female F ₂ mice obtained in Example 1 (homo-deficient type, heterozygous deficient and wild-type) randomly to live a, a marker for breeding establishment morning The female vaginal opening was examined for plugs.
When the plug was confirmed, the female was immediately separated and the period until childbirth was measured.

The results are as shown in FIG. The gestation period of a normal wild-type mouse is 18 to 20 days, and even after one day, the placenta is deteriorated due to the enlargement of the fetus, resulting in stillbirth. On the other hand, in the case of the homo-deficient mouse, the time was extended to about 30 days when the male and female were homo-deficient. However,
No prolongation of gestation was observed when the sex was heterozygous. In addition, it was also confirmed that the effect of the male gene deficiency was not affected by the hetero-deficiency type, and that the homo-deficiency type had no effect if the female was a wild type.

From the above results, it was confirmed that the female of the gene-deficient mouse of the present invention is useful as an infertility model due to female factors. Example 6 (Measurement of Effect of Inhalation Anesthesia in Gene-Deficient Mice) Closed System Observation Cage (40 cm × 60 cm × 40 cm)
Was filled with the inhaled anesthetic sevofuran. Sevofuran concentration was monitored over time and the concentration was constantly adjusted. One wild-type mouse and one gene-deficient mouse are simultaneously placed in this observation cage, and the loss of righting reflex (behavioral reaction to the hands of the experimenter) and analgesia (tail of the mouse) are observed every 5 minutes while maintaining a closed system. The time until a behavioral response to a stimulus with a lightly clipped base was measured. After performing this measurement for 2 hours, the wild-type mouse and the gene-deficient mouse were simultaneously removed from the cage, and the time until the start of walking, the exploratory behavior, and the start of grooming were measured.

The results are as shown in Table 1. In the gene-deficient mouse of the present invention, the disappearance time of the righting reflex and the nociceptive reflex under anesthesia is later than that of the wild-type mouse, and each action start time after awakening is earlier than that of the wild-type mouse except for grooming. From this, it was found that the depth of anesthesia was shallow. Therefore, it was confirmed that the gene-deficient mouse of the present invention is effective as a model for patients with low sensitivity to inhaled anesthetics.

[0032]

[Table 1]

Example 7 (Measurement of Menstrual Cycle in Gene-Deficient Mice) Group rearing of 6 mature female mice in one cage and 1
The animals were acclimated for 3 weeks in a 12-hour light-dark cycle, at room temperature of 23 to 24 ° C. under two kinds of single rearing conditions.
Thereafter, at a certain time every morning, mucosal cells were collected from the vaginal opening using a moistened cotton swab, and based on the type and combination of the cells, proestrus (P), estrus (E), and late estrus-1 (M
1), late estrus-2 (M2) and resting estrus (D).

The results are as shown in FIG. In both group breeding and single breeding, the sexual cycle of wild-type mice was 3 to 5 days, whereas the sexual cycle of the gene-deficient mouse of the present invention was found to be significantly longer, 5 to 9. Therefore, it was confirmed that the gene-deficient mouse of the present invention is effective as a model of irregular physiology.

[0035]

As described above in detail, according to the present invention, the L-PGDS gene has been knocked out, and the deficiency of this synthase causes innate or reactive effects on the central nervous system function, circulatory system function and reproductive function. Gene-deficient mice exhibiting sexual disorders are provided. By using this gene-deficient mouse, 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 a mouse L-PGDS gene (upper), the structure of a mutant sequence in a targeting vector (middle), and the structure of mouse genomic DNA after homologous recombination (lower).

FIG. 2 shows the results of Southern blot analysis of DNA extracted from the tail of each mouse.

FIG. 3 shows the results of Northern blot analysis of mRNA extracted from the brain of each mouse.

FIG. 4 is an analysis result of enzyme activity of L-PGDS extracted from the brain of each mouse.

FIG. 5 shows the results of analgesic sensitivity analysis of a gene-deficient mouse.

FIG. 6 shows the results of counting the number of male offspring of gene-deficient mice.

FIG. 7. L-PG of female wild-type mice during pregnancy
It is an analysis result of the expression level of DS mRNA.

FIG. 8 shows the measurement of the gestation period of a gene-deficient mouse.

FIG. 9 shows the results of measurement of the estrous cycle of wild-type mice and gene-deficient mice.

Claims (11)

[Claims] 1. An early embryo into which a mouse embryonic stem cell in which a lipocalin-type prostaglandin D synthase gene of genomic DNA is homologously recombined with a mutant sequence obtained by replacing exon II with exon V by a neomycin resistance gene is introduced. A descendant of a chimeric mouse generated and produced in an individual in a female mouse, wherein the gene-deficient mouse has a lipocalin-type prostaglandin D synthase gene of germ cell and somatic cell genomic DNA substituted with a mutant sequence . 2. The gene-deficient mouse according to claim 1, wherein both alleles of lipocalin-type prostaglandin D synthase are substituted with a mutant sequence. 3. The gene-deficient mouse according to claim 1, wherein one of alleles of lipocalin-type prostaglandin D synthase is substituted with a mutant sequence. 4. A method for testing the activity of a sleep regulator in an individual, the method comprising administering a candidate substance to the gene-deficient mouse according to claim 1, and measuring the sleep state of the mouse. . 5. A method for testing the activity of an analgesic substance in an individual, comprising administering a candidate substance to the gene-deficient mouse according to claim 1, and measuring the responsiveness of the mouse to a pain stimulus. Test method. 6. A method for testing the activity of a blood coagulation inhibitor in an individual, the method comprising administering a candidate substance to the gene-deficient mouse according to claim 1, and measuring the degree of arteriosclerosis in the mouse. Test method. 7. A method for testing the activity of a substance promoting renal function in an individual, which comprises administering a candidate substance to the gene-deficient mouse according to claim 1, and measuring the renal function of the mouse. Method. 8. A method for testing the intra-individual activity of a fertility-improving substance, comprising administering a candidate substance to the male of the gene-deficient mouse according to claim 1, and subjecting the male mouse to sperm maturation and / or A test method characterized by measuring the degree of reproductive rate of male mice. 9. A method for testing the activity of an infertility-improving substance in an individual, the method comprising the steps of:
A test method comprising administering a candidate substance before or after pregnancy, and measuring the implantation state of a fertilized egg and / or the development state of a fetus in the uterus of the female mouse. 10. A method for testing the activity of an anesthetic substance in an individual, comprising administering a candidate substance to the gene-deficient mouse according to claim 1, and measuring the anesthesia state and / or awake state from the anesthesia of the mouse. A test method characterized by the following: 11. A method for testing the in vivo individual activity of a physiological disorder improving substance, comprising administering a candidate substance to the female of the genetically deficient mouse according to claim 1, wherein the female mouse has an estrous cycle, ovulation number and / or Test method characterized by measuring the amount of various hormones