JP2001299365A - Method for detecting genital abnormality - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting genital abnormalities, to provide a method for screening chemical substance(s) associated therewith, and to provide a method for the tissue culture of the genital tissue associated therewith. SOLUTION: This method for detecting genital abnormality(ies) is characterized by comprising the following steps: the genital tissue in question is cultured in a serumless medium, genes (fibroblast growth factor gene, Msx gene, osteogenetic factor gene, Hox gene, and Shh gene) expressed in the tissue are detected from the tissue under culture, and the presence/absence of the aimed genital abnormality(ies) are detected with the above detection result as indicator.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting genital abnormalities, a method for screening chemical substances, and a method for tissue culture.

[0002]

BACKGROUND ART Developmental abnormalities and malformations have long been studied in various fields as diseases caused by genetic abnormalities.
The occurrence of malformations and malformations has been studied from three perspectives. First, there is a period of malformation. This means a certain period during which malformations can occur when the tissue is affected in the course of development. After the end of the certain period (teratogenic terminal period), it is no longer affected by teratogenesis. Therefore, judgment or recognition of the above-mentioned teratogenic period is important for studying teratogenesis. Secondly, there is a form of malformation. This means that a gene for building a certain tissue is mutated, thereby destroying the primordium, resulting in a malformation. Third, identify the cause of the malformation.
This refers to the identification of physical, chemical, and biological causes, such as physical causes such as radiation damage, chemical causes such as cell necrosis due to lack of oxygen, or organisms such as heart malformation due to viral infection. Is to identify the clinical cause.

[0003] By the way, reproductive abnormalities of animals considered to be caused by chemical substances have been observed in various parts of the world, and it is thought that these abnormalities may be caused by "endocrine disrupters". The starting point was a report from Lake Apopka, Florida, USA, which found that many male crocodile penis had atrophied to 1/4 to 1/2 the normal size. It is considered that these reproductive abnormalities are not problems that can be treated simply as actions of only endocrine disrupters, but that the development and differentiation stages of reproductive organs should be examined at the genetic level.

Conventionally, malformations have been examined at the gene level related to the mechanism of development and differentiation, and certain growth factor genes are involved in the development and differentiation of limb buds (primordial limbs) and lungs. Is known to be important (Development 12
4 (11) 2235-2244, 1997 etc.). In addition, studies using those gene-disrupted mice have been proved to be important.

However, there are many unclear points as to which genes are involved in the occurrence of individual malformations, and this remains as a future subject. In addition, the genes involved in the reproductive organs, particularly the male genitalia (penis) formation or female clitoris formation, have not been elucidated at all, and moreover, the formation process is monitored efficiently, There were no equal methods of determining anomalies. In addition, abnormal development (especially marked external genital abnormalities)
In order to examine how this is caused, only a system that requires a large amount of pregnant mice to administer a chemical substance or the like and determine teratogenicity, which is extremely time-consuming and expensive, has been widely used.

[0006]

An object of the present invention is to provide a method for detecting genital abnormalities, a method for screening chemical substances, and a method for tissue culture.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, as a result of culturing the reproductive organ tissue in vitro using a serum-free medium, the tissue culture system Successfully detected the gene expressed in
The inventor has found that the development process in vitro is the same as that in the mother (in vivo), and has completed the present invention. That is, the present invention is a tissue culture method characterized by culturing a genital tissue in a serum-free medium.

Further, the present invention is characterized in that a genital tissue is cultured in a serum-free medium, a gene expressed in the tissue is detected in the cultured tissue, and a genital abnormality is detected using the obtained detection result as an index. This is a method of detecting genital abnormalities. Further, the present invention, the genital tissue is cultured in the presence of a chemical substance in a serum-free medium, after detecting the gene expressed in the tissue during culture, assay the toxicity of the chemical substance using the obtained detection results as an index A screening method for the chemical substance, characterized in that:

The above-mentioned genes include fibroblast growth factor gene, Msx gene, osteogenic factor gene, Hox gene and
At least one selected from the group consisting of the Shh genes is mentioned, and the genital tissue is an exogenous primordium. In addition, examples of the chemical substance include teratogenic substances such as endocrine disrupting substances. Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION As a means for monitoring the differentiation process typified by extension of the external genitalia in the maternal body, the present inventors cultured embryos (fetuses) in vitro after mating of mammals. We considered monitoring the growth and differentiation process of the cultured tissue under a microscope. At that time, it was thought that by monitoring genes expressed in genital tissues, for example, important genes involved in the formation of limb buds and lungs, the functions of these genes on the development and differentiation of the external genitals could be analyzed.

[0011] Therefore, the present invention provides the same development and differentiation process as that of the mother by removing the reproductive tissue from the embryo (fetal) at a predetermined developmental time and culturing the obtained tissue using a serum-free medium. Can also be observed in vitro. Furthermore, the present invention makes it possible to detect a mutation such as a malformation of a genital tissue by detecting a gene expressed in the tissue under the above culture conditions.

1. Tissue culture Examples of mammals from which tissues are collected include experimental animals such as mice, rats, guinea pigs, rabbits, monkeys, and dogs, with mice being preferred. According to the present invention, after mating an animal, a genital tissue of a fertilized embryo is removed and cultured. In the present invention, the genital tissue means a tissue completely developed and differentiated in the genital organ (male penis, female vagina or clitoris, etc.), as well as a tissue that has not differentiated but is to be differentiated into a genital organ in the future. . However, in the present invention, the primordium portion of the external genitalia (undifferentiated organ during the formation of the external genitalia) is preferable. The outer genital primordia is located in the reproductive nodule and is a tissue rich in mesenchymal cells. Therefore, the primordium is removed from the reproductive nodule under a microscope and cultured.

[0013] The culturing method of the present invention is based on the method of Zoological Science.
11, 847-853, modified from the method described in 1994. That is, the primordium portion is finely scissored (blade 3
４4 mm, thickness of 70 to 100 μm), and then placed on a Millipore filter. Since the Millipore filter is located at the interface (also called interface or interface) between the liquid phase (culture solution) and the gas phase (CO ₂ ), the tissue is cultured at the interface between the gas phase and the liquid phase. In the present invention, for the purpose of monitoring the effects of humoral factors and chemicals,
Use serum-free medium.

The medium is not particularly limited as long as it is a medium for culturing animal tissues or cells.
Jb medium, Eagle's minimum essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), Moor et al. RPMI-1640 medium,
RITC80-7 culture solution of Yamane et al., RITC62-8 culture solution, synthetic culture solution of Ham F12, L-15 culture solution of Leibovitz, synthetic culture solution NCTC109 of Evans et al., Synthetic culture solution of Parker et al. CMRL-1066, McCo
y5a culture, Williams culture E, Katsuta et al. DM-160
Culture solution, SFM-101 culture solution and the like.

The filter preferably has a pore size of 0.2 to 0.8 μm, and more preferably 0.4 μm. The culture can be performed under the same conditions as those for culturing normal animal tissues or cells. For example, at a temperature of 37 ° C. and 5% CO ₂ , usually within 6 days, preferably 1 to
Incubate for 3 days. It is also possible to change the O ₂ concentration in the range of 10% to 95%.

2. Detection of Gene Expression The gene to be detected in the present invention is not particularly limited as long as it is a gene expressed in external genital tissues. For example, a gene encoding a fibroblast growth factor (Fgf gene), a mouse Msx-1 gene containing a homeobox and involved in the regulation of development (Alasdair, M. et al., De
velopment, 111, 269-285 (1991)), the Bmp-4 gene encoding a bone morphogenetic factor (Winnier, G. et al., Genes Dev.
9, 2105-2116 (1995)), involved in the formation of limbs (limb buds)
Hox gene (for example, Hoxd-13 gene: Dolle, P. et al.,
MOD 36, 3-13 (1991)), Shh gene (Echelar encoding sonic hedgehog, which is important for limb (limb bud) and nervous system formation)
d, Y. et al., Cell, 75 (7) 1417-1430 (1993)).

The Fgf gene is thought to be involved in the formation of limb buds and lungs, and forms a family from Fgf-1 to 20. In the present invention, any of these can be used, but Fgf-8 (Tanaka, A. et al., Proc. Nat.
l. Acad. Sci. USA 89 (19) 8928-8932 (1992) or Fgf
-10 (Tagashira, S. et al., Gene, 197 (1-2) 399-404 (1
997)) is preferred.

In the tissues in culture, that is, tissues (early epithelium, mesenchyme, etc.) in the early stage of tissue culture (1 day from the start of culture), the middle stage (1 to 3 days), and the late stage (3 to 6 days), Detection is performed by gene expression analysis. Detection of gene expression
itu RNA expression (in situ hybridization kit: A
Techniques such as mbion, R & D, Takara, Boehringer, RT-PCR (Takara RNA RT-PCR kit: Takara Shuzo, etc.), Northern blotting and the like are used. However, the present invention is not limited to the above method. These techniques are known to those skilled in the art.

The oligonucleotide used for the detection of the above gene expression can be appropriately designed and synthesized according to the type of the gene to be detected. In addition, a commercially available chemical synthesizer (manufactured by PerkinElmer) or the like can be used for oligonucleotide synthesis.

When the expression of each gene is detected, any of the full-length sequence or partial sequence of each gene (see below) or a sequence obtained by adding a sequence of an untranslated region to these sequences can be used as a probe. . Also,
These genes are used alone or in combination of two or more.

Fgf-8 gene (SEQ ID NO: 1) Fgf-10 gene: (SEQ ID NO: 2) Shh gene: (SEQ ID NO: 3) Bmp-4 gene: (SEQ ID NO: 4) Msx-1 gene: (SEQ ID NO: 5)

For example, when detecting the Fgf-8 gene, the sequence from the 160th to the 997th nucleotide (SEQ ID NO: 6) of the nucleotide sequence shown in SEQ ID NO: 1 is used.
In the case of detecting the Shh gene, the 1st to 630th sequence of the nucleotide sequence shown in SEQ ID NO: 3 includes the 1st to 870th sequence of the nucleotide sequence shown in SEQ ID NO: 3, or the sequence of the untranslated region upstream of the sequence. When detecting the Bmp-4 gene, the 215th to 1731st sequences of the nucleotide sequence shown in SEQ ID NO: 4, and when detecting the Msx-1 gene, the 733th to 733th nucleotides of the nucleotide sequence shown in SEQ ID NO: 5
The 1328th sequence or the sequence containing the sequence of the untranslated region downstream of the sequence can be exemplified as the probe.

3. In-vitro and in-vivo comparison of genital extension It is performed by a method of confirming that the stage is a generation stage. Also, confirm that tissues cultured in vitro are at the same developmental stage. Then, for the tissue observed in vivo and the tissue observed in vitro, the expression of the gene was confirmed and the morphology of the external genital extension was observed, and the results in vitro and the results in vivo were correlated. Check. When examining the expression of the Bmp-4 gene, Fgf-8 gene or Fgf-10 gene,
Good correlation can be obtained for tissue development and differentiation. In the present invention, since in vitro results and in vivo results have a correlation in normal tissues, this data can be used for detection of genital abnormalities (eg, malformations).

That is, the tissue to be detected is cultured by the above-described tissue culture method, and the target gene is detected by the above-described gene detection method. When the obtained detection result is different from the behavior of the normal tissue, that is, when the expression of the target gene is not recognized, or when the obtained pattern shows a pattern different from the original pattern (eg, ectopic expression), the tissue to be detected is used. Can be determined to have a high probability of becoming malformed.
On the other hand, if the obtained detection result is similar to the behavior of normal tissue, or if the expression intensity of the target gene is equal to that of normal, it can be determined that the tissue to be detected has a low probability of becoming malformed. it can.

4. Chemical Substance Screening In the present invention, when a chemical substance is present in culturing a genital tissue in a serum-free medium, the effect of the chemical substance on the development and differentiation of the genital tissue is assayed (monitored). Can be. That is, it is possible to know how toxic a chemical substance is, using an abnormality in the expression of a gene expressed in genital tissues as an index. As a result, the method of the present invention is used for teratogenicity tests of chemical substances such as drugs,
It can be used for screening of the chemical substance.

Chemicals include endocrine disrupting substances called so-called environmental hormones (for example, dioxin,
In addition to bisphenol A), organic substances (diethylstilbestrol (DES)), metals (organotin, organomercury), cancer-inducing agents (eg, methylcholanthrene) and the like can be mentioned. The above-mentioned chemical substance is present in the medium in an amount of about 1 × 10 ⁻¹⁰ mg to about ¹⁰ mg (about 10 ⁻⁹ M to about 10 ⁻⁴ to 10 ⁻³ M) per unit amount (1 ml), and the same method as the culture method is used. Culture the genital tissue. Then, the expression of the gene is detected in the early, middle and late culture stages.

In order to test the toxicity of a chemical substance from the detection result of gene expression, the following criteria are used. In other words, when the gene expression is similar to the standard determined as abnormal, the target chemical substance is determined to be toxic. For example, when the gene expression pattern detected by the probe used is enlarged (including an ectopic pattern), reduced, or the gene expression is increased, attenuated or eliminated, or Or, the case where the gene expression of the combination of and or was shown. On the other hand, when the gene expression is similar to the standard for determining that the expression is normal, it can be determined that there is no or low toxicity. For example, there is a case where the gene expression pattern in the test group is compared (overlaid) with the pattern in the control group and no significant change is observed.

The test is carried out by changing the amount of the chemical substance present in the medium. If the test substance is determined to be non-toxic or low-toxic at a low concentration and determined to be toxic at a high concentration with respect to the reference concentration relating to the toxicity of the test substance, the test substance is tested. The level of toxicity of the test chemical can be known, and the test chemical is selected.

In general, in a toxicity test using pregnant mice, rats, and the like, the number of animals used and the test substance are related to one substance per animal (one administration condition). In the present invention, a plurality (about 10 to 12) fetuses of one pregnant animal can be used. Therefore, the use of a serum-free organ culture system allows one pregnant animal
Zero or more types of substance administration conditions can be set, and those substances can be assayed (about 10-fold test efficiency can be obtained). Therefore, in the present invention, an initial toxicity assay can be performed, and a more detailed toxicity assay can be performed in a conventional individual animal system.

[0030]

The present invention will be described more specifically with reference to the following examples. However, the technical scope of the present invention is not limited to these examples. Example 1 Investigation of External Genital Development and Differentiation Process in Tissue Culture System In this example, external genital development and differentiation in the maternal body were the same as external genital development and differentiation in the tissue culture system of the present invention. Was examined, ie, whether the development and differentiation of tissues in vivo correspond in vitro.

After mating a mouse (ICR strain, 10 weeks old), a reproductive nodule of a fertilized embryo was collected. 0.4 μm pore Millipore filter (HTTP) coated with 5% gelatin solution
Was placed in a tissue culture dish. Culture was carried out such that the outer genital primordia was located at the interface between the medium (liquid phase) and the gas phase.

As a medium, a serum-free medium without FBS was used. 0.1 mg / ml ascorbic acid and 10 mM Hepes (pH 7.
4) In BGJb medium containing
The culture was grown to the stage corresponding to the tissue when it grew in the (maternal) from about 10 to 14 days. Using the tissue in culture, a probe specific to the sequence of the Fgf-8 gene or Fgf-10 gene was used, and an in situ RNA expression kit (Am
Bion) was used to detect the gene expression of Fgf-8 and -10. As a control, the gene expression pattern was similarly analyzed for the tissues at the same time in the mother. Probe labels were prepared by labeling with digoxigenin (DIG).

Probe sequence: For detecting Fgf-8 gene: SEQ ID NO: 6 (positions 160 to 997 of the nucleotide sequence shown in SEQ ID NO: 1) Detection for Fgf-10 gene: SEQ ID NO: 2 (positions 1 to 630) As a result, the tissue culture system of the present invention morphologically showed a developmental process almost coincident with a certain time after fertilization in the mother. And the tissue culture system showed a developmental process almost the same as that after fertilization in the mother (FIG. 1).

In FIG. 1, the developmental image of the external genital primordia 12.5 days after fertilization in vivo (maternal body) (left panel (E)) is shown, and the in vitro cultured tissue is collected and cultured for 1 day. It was found that the expression level of the Fgf-8 gene was consistent with the development image (right panel (I)).

FIG. 2 shows that Fgf-8 and Fgf-
It is the result of examining the distribution of expression of 10 genes. The Fgf-8 gene (right panel (K): 11.5 days after fertilization) is highly expressed in the apical part (epithelial epithelium at the tip of the urethra), and the Fgf-10 gene (left panel (H): 11.5 days after fertilization) is near the tip It was found that the expression was high in the mesenchymal tissue. Fg in the above
When the expression of the f family gene was confirmed in vitro and in vivo, both showed similar expression patterns.

Example 2 Examination of the Effect of FGF-8 Protein on External Genital Elongation 1 mg / ml of a commercially available FGF-8 protein (manufactured by R & D) was adsorbed on beads of Sigma, and this was used in Example 1. It was added to the indicated tissue culture system and morphological observation was performed. As a result, external genital extension was observed when FGF-8 protein was added, and when a commercially available (manufactured by R & D) anti-FGF-8 protein antibody was added in the same manner as described above, external genital extension was suppressed. (FIGS. 3 and 4).

In FIG. 3, the external genital primordium of the control group (left panel (c)) shows no elongation of the external genitalia, whereas the tissue to which FGF-8 protein was added (right panel (right panel)). In d)), morphological extension of the external genitalia was clearly observed. In addition, in FIG. 4, the addition of anti-FGF-8 antibody suppressed the growth of the external genitalia,
The f family gene was shown to be essential. In addition, the vertical axis of FIG. 4 indicates the relative external genital elongation (measured the distance from the genital epithelium of the genital tip to the developing foreskin at the base,
The relative ratio (%) with the normal group taken as 100%) is shown.

Example 3 DES for Bmp-4 Gene Expression
Examination of effects of mice After mating mice (ICR strain, 10 weeks old), reproductive nodules of fertilized embryos were collected. A 0.4 μm pore Millipore filter (HTTP) coated with a 5% gelatin solution was placed on the tissue culture dish. Culture was carried out such that the outer genital primordia was located at the interface between the medium (liquid phase) and the gas phase.

As a medium, a serum-free medium without FBS was used. 0.1 mg / ml ascorbic acid and 10 mM Hepes (pH 7.
In BGJb medium containing 4), the embryonic tissue was cultured in vivo (mother) to a stage corresponding to the tissue that had developed to about 13.5 days. DE in the above stage tissue culture medium
S was added to 1 μM or 10 μM, and after 24 hours, the expression of Bmp-4 gene, a bone morphogenetic gene, was detected. The gene expression was detected using an in situ RNA expression kit (Ambion) using a probe specific to the Bmp-4 gene sequence. Probe labels were prepared by labeling with digoxigenin (DIG).

Sequence of probe for detecting Bmp-4 gene: Sequences at positions 215 to 1731 of the nucleotide sequence shown in SEQ ID NO: 4 As a control, the gene expression pattern of a tissue obtained by adding only DMSO instead of DES to the medium was used. Was analyzed. As a result, in the tissue culture system to which DES was added,
In particular, when 10 μM was added, the expression of the BMP-4 gene was significantly suppressed (FIG. 5). In FIG. 5, A is a control tissue, and B is a control tissue.
Is the developmental image of genital primordia when 1 μM DES was added, C is D
1 shows a developmental image of external genital primordia when 10 μM of ES was added.

[0041]

According to the present invention, a method for detecting genital abnormalities,
A method for screening a chemical substance and a method for tissue culture are provided. In addition, by using the present invention to detect the expression of a plurality of genes, it is possible to monitor the effects of various teratogenic factors and endocrine disrupting substances (environmental hormones). Furthermore, the method of the present invention detects how chemicals, such as endocrine disruptors, affect the external genitalia formation program in mammals, and any chemicals, synthetics are toxic to the reproduction of each species. This is useful in that it can be evaluated.

[0042]

[Sequence List] SEQUENCE LISTING <110> Kumamoto technopolis foundation <120> Method of detecting genital abnormality <130> P01-0082 <150> JP2000-36084 <151> 2000-02-15 <160> 6 <170> PatentIn Ver. 2.0 <210> 1 <211> 997 <212> DNA <213> Mus musculus <400> 1 cgcgcgcggc gagcacgaca ttccaccgga cccgccgagc cgcgtcggga tagccgctgg 60 cctcccgcac cccgacctcc ctcagcctcc gcaccttcgg cttgtccccc cgcggcctcc 120 agtgggacgg cgtgaccccg ctcgggctct cagtgctccc ggggccgcgc gccatgggca 180 gcccccgctc cgcgctgagc tgcctgctgt tgcacttgct ggttctctgc ctccaagccc 240 aggtaactgt tcagtcctca cctaatttta cacagcatgt gagggagcag agcctggtga 300 cggatcagct cagccgccgc ctcatccgga cctaccagct ctacagccgc accagcggga 360 agcacgtgca ggtcctggcc aacaagcgca tcaacgccat ggcagaagac ggagacccct 420 tcgcgaagct cattgtggag accgatactt ttggaagcag agtccgagtt cgcggcgcag 480 agacaggtct ctacatctgc atgaacaaga aggggaagct aattgccaag agcaacggca 540 aaggcaagga ctgcgtattc acagagatcg tgctggagaa caactacacg gcgctgcaga 600 acgccaagta cgagggctgg tacatggcct ttacccgcaa gg gccggccc cgcaagggct 660 ccaagacgcg ccagcatcag cgcgaggtgc acttcatgaa gcgcctgccg cggggccacc 720 acaccaccga gcagagcctg cgcttcgagt tcctcaacta cccgcccttc acgcgcagcc 780 tgcgcggcag ccagaggact tgggccccgg agccccgata ggcgctcgcc cagctcctcc 840 ccacccagcc ggccgaggaa tccagcggga gctcggcggc acagcaaagg ggaggggctg 900 gggagctgcc ttctagttgt gcatattgtt tgctgttggg tttttttgtt ttttgttttt 960 tgtttttgtt ttttgttttt taaacaaaag agaggcg 997 <210> 2 <211> 630 <212> DNA <213> Mus musculus <400> 2 atgtggaaat ggatactgac acattgtgcc tcagcctttc cccacctgcc gggctgctgt 60 tgctgcttct tgttgctctt tttggtgtct tcgttccctg tcacctgcca agctcttggt 120 caggacatgg tgtcacagga ggccaccaac tgctcttctt cctcctcgtc cttctcctct 180 ccttccagtg cgggaaggca tgtgcggagc tacaatcacc tccaaggaga tgtccgctgg 240 agaaggctgt tctccttcac caagtacttt ctcacgattg agaagaacgg caaggtcagc 300 gggaccaaga atgaagactg tccgtacagt gtcctggaga taacatcagt ggaaatcgga 360 gttgttgccg tcaaagccat caacagcaac tattacttag ccatgaacaa gaaggggaaa 420 ctctatggct caaaagagtt taacaacgac tgta agctga aagagagaat agaggaaaat 480 ggatacaaca cctatgcatc ttttaactgg cagcacaatg gcaggcaaat gtacgtggca 540 ttgaatggaa aaggagctcc caggagagga caaaaaacaa gaaggaaaaa cacctctgct 600 cacttcctcc ccatgacgat ccaaacatag 630 <210> 3 <211> 1314 <212> DNA <213> Mus musculus <400> 3 atgctgctgc tgctggccag atgttttctg gtgatccttg cttcctcgct gctggtgtgc 60 cccgggctgg cctgtgggcc cggcaggggg tttggaaaga ggcggcaccc caaaaagctg 120 acccctttag cctacaagca gtttattccc aacgtagccg agaagaccct aggggccagc 180 ggcagatatg aagggaagat cacaagaaac tccgaacgat ttaaggaact cacccccaat 240 tacaaccccg acatcatatt taaggatgag gaaaacacgg gagcagaccg gctgatgact 300 cagaggtgca aagacaagtt aaatgccttg gccatctctg tgatgaacca gtggcctgga 360 gtgaagctgc gagtgaccga gggctgggat gaggacggcc atcattcaga ggagtctcta 420 cactatgagg gtcgagcagt ggacatcacc acgtccgacc gggaccgcag caagtacggc 480 atgctggctc gcctggctgt ggaagcaggt ttcgactggg tctactatga atccaaagct 540 cacatccact gttctgtgaa agcagagaac tccgtggcgg ccaaatccgg cggctgtttc 600 ccgggatccg ccaccgtgca cctggagcag gg cggcacca agctggtgaa ggacttacgt 660 cccggagacc gcgtgctggc ggctgacgac cagggccggc tgctgtacag cgacttcctc 720 accttcctgg accgcgacga aggcgccaag aaggtcttct acgtgatcga gacgctggag 780 ccgcgcgagc gcctgctgct caccgccgcg cacctgctct tcgtggcgcc gcacaacgac 840 tcggggccca cgcccgggcc aagcgcgctc tttgccagcc gcgtgcgccc cgggcagcgc 900 gtgtacgtgg tggctgaacg cggcggggac cgccggctgc tgcccgccgc ggtgcacagc 960 gtgacgctgc gagaggagga ggcgggcgcg tacgcgccgc tcacggcgca cggcaccatt 1020 ctcatcaacc gggtgctcgc ctcgtgctac gctgtcatcg aggagcacag ctgggcacac 1080 cgggccttcg cgcctttccg cctggcgcac gcgctgctgg ccgcgctggc acccgcccgc 1140 acggacggcg ggggcggggg cagcatccct gcagcgcaat ctgcaacgga agcgaggggc 1200 gcggagccga ctgcgggcat ccactggtac tcgcagctgc tctaccacat tggcacctgg 1260 ctgttggaca gcgagaccat gcatcccttg ggaatggcgg tcaagtccag ctga 1314 <210> 4 <211> 1785 <212> DNA <213> Mus musculus <400 > 4 atgtcttacg gtcaaggggc atccgagctg agagacccca gcctaagacg ccgctgcgct 60 gcaacccagc ctgagtatct ggtctccgtc cctgatggga ttctcgtcta aaccgtcttg 120 gagcctcgac gagtccagtc tctggccctc gaccaggttc attgcagctt tctagaggtc 180 cccagaagca gctgctggcg agcccgcttc tgcaggaacc aatggagcca ttccgtagtg 240 ccattcggag cgacgcactg ccgcagcttc tctgagcctt tccagcaagt ttgttcaaga 300 ttggctccca agaatcatgg actgttatta tatgccttgt tttctgtcaa gacaccatga 360 ttcctggtaa ccgaatgctg atggtcgttt tattatgcca agtcctgcta ggaggcgcga 420 gccatgctag tttgatacct gagaccggga agaaaaaagt cgccgagatt cagggccacg 480 cgggaggacg ccgctcaggg cagagccatg agctcctgcg ggacttcgag gcgacacttc 540 tacagatgtt tgggctgcgc cgccgtccgc agcctagcaa gagcgccgtc attccggatt 600 acatgaggga tctttaccgg ctccagtctg gggaggagga ggaggaagag cagagccagg 660 gaaccgggct tgagtacccg gagcgtcccg ccagccgagc caacactgtg aggagtttcc 720 atcacgaaga acatctggag aacatcccag ggaccagtga gagctctgct tttcgtttcc 780 tcttcaacct cagcagcatc ccagagaatg aggtgatctc ctcggcagag ctccggctct 840 ttcgggagca ggtggaccag ggccctgact gggaacaggg cttccaccgt ataaacattt 900 atgaggttat gaagccccca gcagaaatgg ttcctggaca cctcatcaca cgactactgg 960 acaccagact agtccatca c aatgtgacac ggtgggaaac tttcgatgtg agccctgcag 1020 tccttcgctg gacccgggaa aagcaaccca attatgggct ggccattgag gtgactcacc 1080 tccaccagac acggacccac cagggccagc acgtcagaat cagccgatcg ttacctcaag 1140 ggagtggaga ttgggcccaa ctccggcccc tcctggtcac ttttggccat gatggccggg 1200 gccatacctt gacccgcaga agggccaaac gtagtcccaa gcatcaccca cagcggtcca 1260 ggaagaagaa taagaactgc cgtcgccatt cactatacgt ggacttcagt gacgtgggct 1320 ggaatgattg gattgtggcc ccacccggct accaggcctt ctactgccac ggggactgtc 1380 cctttccact ggctgatcac ctcaactcaa ccaaccatgc cattgtgcag accctagtca 1440 actctgttaa ttctagtatc cctaaggcct gttgtgtccc cactgaactg agtgccattt 1500 ccatgttgta cctggatgag tatgacaagg tggtgttgaa aaattatcag gagatggtgg 1560 tagaggggtg tggatgccgc tgagatcagg cagtccggag ggcagacaca cacacacaca 1620 cacacacaca cacacacaca cacacacacg ttcccattca accacctaca cataccacaa 1680 actgcttccc tatagctgga cttttatctt aaccttgacc gtaaagacat gaattcgagc 1740 tcggtacccg gggatcctct agagtcgacc tgcaggcatg caagc 1785 <210> 5 <211> 1810 <212> DNA <213> Mus mus culus <400> 5 ggaacccagg agctcgcaga agccggtcag gagctcgcag aagccggtcg cgctcccagc 60 ctgcccgaaa cccatgatcc agggctgtct cgagctgcgg ctggaggggg ggtccggctc 120 tgcatggccc cggctgctgc tatgacttct ttgccactcg gtgtcaaagt ggaggactcc 180 gccttcgcca agcctgctgg gggaggcgtt ggccaagccc ccggggctgc tgcggccacc 240 gcaaccgcca tgggcacaga tgaggagggg gccaagccca aagtgcccgc ttcactcctg 300 cccttcagcg tggaggccct catggccgat cacaggaagc ccggggccaa ggagagcgtc 360 ctggtggcct ccgaaggggc tcaggcagcg ggtggctcgg tgcagcactt gggcacccgg 420 cccgggtctc tgggcgcccc ggatgcgccc tcctcgccgc ggcctctcgg ccatttctca 480 gtcggaggac tcctcaagct gccagaagat gctctggtga aggccgaaag ccccgagaaa 540 ctagatcgga ccccgtggat gcagagtccc cgcttctccc cgcccccagc cagacggctg 600 agtcccccag catgcaccct acgcaagcac aagaccaacc gcaagcccag gacgcctttc 660 accacagctc agctgctggc tctggagcgc aagttccgcc agaagcagta cctgtctatt 720 gccgagcgcg cggaattctc cagctcgctc agcctcaccg agacccaggt gaagatctgg 780 ttccagaacc gtcgcgctaa ggccaagaga ctgcaggagg cggagctgga gaagctgaag 840 atggccg cga aacccatgtt gccgcctgct gccttcgctc tcttttcctc ttggcggtcc 900 tgcagcggtg gctgcagctg cgggcgcctc actctacagt gcctctggcc ctttccagcg 960 cgccgcgctg cctgtagcgc ccgtgggact ctacaccgcc catgtaggct acagcatgta 1020 ccacctgact taggtgggtc cagagtcacc tccctgtggt gccatcccct ccccagccac 1080 ctctttgagc agagcagcgg gagtccttcc taggaagctc tgctgcccta taccacctgg 1140 tcccttctct taaacccctt gctacacact tcctcctggt tgtcgcttcc taaaccttcc 1200 tcatctgacc ccttctggga agaaaaagaa ttggtcggaa gatgttcagg tttttcgagt 1260 tttttctaga tttacatgcg caagttataa aatgtggaaa ctaaggatgc agaggccaag 1320 agatttatcc gtggtcccca gcagaattag aggctgaagg agaccagagg ccaaaaggac 1380 tagaggccat gagactccat cagctgcttc cggtcctgaa accaggcagg acttgcacag 1440 agaaattgct aagctaatcg gtgctccaag agatgagccc agccctatag aaagcaagag 1500 cccagctcct tccactgtca aactctaagc gctttggcag caaagcattg ctctgagggg 1560 gcagggcgca tgctgctgct tcaccaaggt aggttaaaga gactttccca ggaccagaaa 1620 aaaagaagta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa caaatctgtt ctattaacag 1680 tacattttcg tggc tctcaa gcatcccttt tgaagggact ggtgtgtact atgtaatata 1740 ctgtatattt gaaattttat tatcatttat attatagcta tatttgttaa ataaattaat 1800 tttaagctac 1810 <210> 6 <211> 828 <212> DNA <213> Mus musculus <400> 6 cgccatgggc agcccccgct ccgcgctgag ctgcctgctg ttgcacttgc tggttctctg 60 cctccaagcc caggtaactg ttcagtcctc acctaatttt acacagcatg tgagggagca 120 gagcctggtg acggatcagc tcagccgccg cctcatccgg acctaccagc tctacagccg 180 caccagcggg aagcacgtgc aggtcctggc caacaagcgc atcaacgcca tggcagaaga 240 cggagacccc ttcgcgaagc tcattgtgga gaccgatact tttggaagca gagtccgagt 300 tcgcggcgca gagacaggtc tctacatctg catgaacaag aaggggaagc taattgccaa 360 gagcaacggc aaaggcaagg actgcgtatt cacagagatc gtgctggaga acaactacac 420 ggcgctgcag aacgccaagt acgagggctg gtacatggcc tttacccgca agggccggcc 480 ccgcaagggc tccaagacgc gccagcatca gcgcgaggtg cacttcatga agcgcctgcc 540 gcggggccac cacaccaccg agcagagcct gcgcttcgag ttcctcaact acccgccctt 600 cacgcgcagc ctgcgcggca gccagaggac ttgggccccg gagccccgat aggcgctcgc 660 ccagctcctc cccacccagc cggccgagga atccagcggg agctcggcgg cacagcaaag 720 gggaggggct ggggagctgc cttctagttg tgcatattgt ttgctgttgg gtttttttgt 780 tttttgtttt ttgttttttttttttttttttttt ttaaacaaaa gagaggcg 828

[Brief description of the drawings]

FIG. 1. In vitro expression of Fgf-8 gene in external genital primordia
4 is a photograph showing the results of comparison between vo and in vitro.

FIG. 2 is a photograph showing the results of examining the distribution of Fgf-8 and Fgf-10 gene expression in vivo.

FIG. 3 is a photograph showing the effect of FGF-8 protein administration on external genital extension.

FIG. 4. FGF-8 protein and anti-F against external genital extension
It is a figure which shows the effect of GF-8 protein antibody administration.

FIG. 5. DES for Bmp-4 gene expression in external genitalia.
FIG. 9 is a diagram showing the results of a test on the effect of the above.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 E

Claims (9)

[Claims] 1. A tissue culture method comprising culturing a genital tissue in a serum-free medium. 2. The genital tissue is an external genital primordium.
The method for tissue culture according to the above. 3. A genital tissue is cultured in a serum-free medium, and a gene expressed in the cultured tissue is detected.
A method for detecting genital abnormalities, comprising detecting genital abnormalities using the obtained detection result as an index. 4. The gene is a fibroblast growth factor gene,
The detection method according to claim 3, wherein the detection method is at least one selected from the group consisting of an Msx gene, a bone morphogenetic gene, a Hox gene, and a Shh gene. 5. The genital tissue is an external genital primordium.
Or the detection method according to 4. 6. A method of culturing a genital tissue in a serum-free medium in the presence of a chemical substance, detecting a gene expressed in the tissue during the culture, and assaying the toxicity of the chemical substance using the obtained detection result as an index. A method for screening a chemical substance as described above. 7. The screening method according to claim 6, wherein the chemical substance is an endocrine disrupting substance. 8. The gene is a fibroblast growth factor gene,
8. The screening method according to claim 6, wherein the screening method is at least one selected from the group consisting of an Msx gene, a bone morphogenetic gene, a Hox gene, and a Shh gene. 9. The genital tissue according to claim 6, wherein the genital tissue is an external genital primordium.
The screening method according to any one of claims 1 to 8.