JPH11318468A - New protein participating in differentiation of carnial nerve tissue cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein which is composed of a protein that comprises a specific amino acid sequence and possesses activity inducing differentiation in the region of carnial nerves and is useful in the elucidation of differentiation mechanism in the region of carnial nerves or the prevention and treatment for disease caused by abnormal carnial nerve tissues. SOLUTION: This new protein is composed of the amino acid sequence of the formula or the amino acid sequence in which one or plural amino acids are substituted, lacked or added on the amino acid sequence in the protein, possesses activity inducing differentiation in carnial nerve tissues and is useful in the elucidation of differentiation mechanism in carnial nerve tissues and consequently the prevention and treatment for disease caused by abnormal carnial nerve tissues. This protein is obtained by initially separating mRNA from the fraction of central nervous system in mouse embryos by a conventional method, making a plasmid cDNA library by the use of this mRNA, cloning this by carrying out PCR(polymerase chain reaction) with the primer consisting of a partial sequence and finally integrating the resultant gene into a vector to express in host cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel protein involved in the differentiation of brain nerve tissue cells and its gene.

[0002]

BACKGROUND OF THE INVENTION Many genes are involved in the process of morphogenesis during embryonic development, and these genes cause cells to differentiate and form specific tissues or organs. Abnormalities in these genes involved in morphogenesis can result in lethality during development or insufficient formation of specific tissues or organs, resulting in functionally abnormal individuals.

[0003] In particular, cerebral nervous system tissue cells are control towers for controlling life activities and are one of the most important organs for life activities. The basic ability of living organisms to respond to external stimuli and respond appropriately to them, not to mention the advanced mental activities found in humans, is based on highly developed brain and nervous system tissues. Depends on the work of For this reason, if the formation or function of the cerebral nervous system tissue becomes abnormal, a great hindrance to maintaining normal life activity will occur. For this reason, it is very important to analyze the formation of the cerebral nervous system tissue to open the way to prevention and treatment of diseases caused by abnormalities in the cerebral nervous system tissue. In analyzing the formation of the cerebral nervous system tissue, it is an important step to first isolate a gene involved in the formation of the cerebral nervous system tissue and identify its function.

As a gene involved in the formation of a cerebral nervous system tissue, Lhx1 (W. Shawlot and RR Behringer, 1995, Nature, 374), which has been reported so far to be knocked out, results in loss of the head. 425-430), head,
Mice deficient in this gene, which is essential for medial mesodermal formation, have Oxt2 (IM
atsuo et al., 1995, Genes Dev., 9: 2646-2658), and Wnt-1 (APMcMaho
n and A. Bradley, 1990, Cell, 62: 1073-1085).

[0005] However, the differentiation mechanism of the cerebral nervous tissue has not yet been fully elucidated, and the isolation and analysis of a new gene involved in the formation of the cerebral nervous system tissue has been difficult. It is desired for elucidation, and further prevention and treatment of diseases caused by abnormalities of the cerebral nervous system tissue.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel protein involved in the formation of a nervous system tissue and a DNA encoding the protein. Another object of the present invention is to provide a vector into which the DNA is inserted, a transformant carrying the DNA, and a method for producing a recombinant protein using the transformant. Still another object of the present invention is to provide an antibody and an oligonucleotide used for detection and isolation of the protein and the DNA. Still another object of the present invention is to provide a transgenic mouse into which the DNA has been introduced and a knockout mouse in which the DNA has been modified or destroyed.

[0007]

Means for Solving the Problems As a method for isolating an unidentified gene involved in a specific function, hybridization using a sequence of a gene known to have the specific function is known. And a method utilizing the polymerase chain reaction (PCR). However, this method has a disadvantage that a gene having low homology to a known gene cannot be isolated. On the other hand, as a method for isolating an unknown gene involved in a specific function irrespective of homology with a known gene, a gene trap (GT) method is used (A. Gossler et al., 1989). ,
Science 244: 463-465; Z. Chen et al., Genes Dev. 8: 22
93-2301; J. De Gregori et al., 1994, Genes Dev. 8: 265-
276; T. Takeuchi et al., 1995, Genes Dev. 9: 1211-122.
2). In the gene trap method, a vector (trap vector) containing an appropriate marker gene in a state lacking a promoter sequence was maintained in an undifferentiated state in embryonic stem cells (cells derived from the inner mass of a fertilized egg at the blastocyst stage). It is a cell that can be cultured as is.It is widely used for the production of transgenic animals. Hereinafter, it is integrated into the genome of "ES cells".
When this vector is integrated on a specific gene in the genome, the marker gene is expressed according to the expression pattern, and the gene is isolated using this as an index. However, this method generally has a disadvantage that only genes expressed in ES cells can be searched.

Therefore, the present inventors have developed a trap vector independently in order to isolate an unknown gene that is expressed at a late stage such as organogenesis during embryo development (FIG. 1), Screening for genes involved in organ formation in E. coli. As a result, during the mouse embryonic development process, we succeeded in isolating novel genes that are expressed especially in the commissure nerves and digestive tract. The isolated gene has been suggested to be a tumor suppressor gene in rat DA
PRDC, which has low homology with the translation products of the `` N '' gene and the Xenopus `` Cerberus '' gene, which is known to form secondary embryos
(protein related to DAN and cerberus).
The present inventors injected “PRDC” mRNA into Xenopus laevis embryos and examined the effects on embryo morphology. As a result, it was found that secondary formation of the head and body axis was induced in Xenopus embryos (FIG. 7). From these facts, the present inventors have found that the “PRDC” protein
It is thought that it is particularly involved in the formation of cranial nerves during mouse embryonic development.

[0009] The present invention relates to a novel protein involved in the differentiation of brain nerve tissue cells, a gene encoding the protein, and use thereof, and more specifically, (1)
A protein comprising the amino acid sequence of SEQ ID NO: 1, or a protein comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence and having an activity of inducing differentiation of brain nerve tissue (2) a protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 2, which has an activity of inducing differentiation of brain nerve tissue, (3) (1) or (2) DNA encoding the protein of (4), a vector into which the DNA of (3) has been inserted, (5) a transformant capable of expressing the DNA of (3) in an expressible manner, (6)
Culturing the transformant according to (5),
The method for producing a protein according to (1) or (2),
(7) an antibody that binds to the protein according to (1) or (2), (8) specifically hybridizes with a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2, and has a chain length of at least 15 nucleotides DNA, (9) SEQ ID NO: 1
Encodes a protein consisting of the amino acid sequence of
A transgenic mouse that holds the DNA so that it can be expressed,
(10) A knockout mouse in which a genomic DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 has been modified or destroyed.

[0010]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel protein involved in the differentiation of brain nerve tissue cells. The nucleotide sequence of the cDNA encoding the protein named "PRDC" contained in the protein of the present invention is shown in SEQ ID NO: 2, and the amino acid sequence of the protein is shown in SEQ ID NO: 1.

The "PRDC" cDNA isolated by the present inventors is expressed in the spinal cord of mouse embryos, particularly in a group of commissure nerves (Example 3).
And 8) In addition, injection of "PRDC" mRNA into Xenopus embryos induces secondary head formation (Example 11). From these facts, it is considered that "PRDC" in vivo is mainly involved in induction of differentiation of the cranial nervous system. For this reason, the “PRDC” protein and its gene, as well as compounds that regulate the function of the “PRDC” protein, can be applied to the treatment of diseases related to the cerebral nervous system.

The protein of the present invention can be prepared by a method known to those skilled in the art as a recombinant protein prepared using a gene recombination technique or as a natural protein. In the case of a recombinant protein, for example, a DNA encoding the protein of the present invention (for example, a DNA having the nucleotide sequence of SEQ ID NO: 2) is inserted into an appropriate expression vector, and this is introduced into a host cell. It can be prepared by a method such as purification from a transformant. In addition, if it is a natural protein, for example, a column in which an antibody obtained by immunizing a small animal with the prepared recombinant protein is prepared,
It can be prepared by a method such as performing affinity chromatography using an extract on a tissue or cell (eg, commissure nerve tissue) expressing the protein of the present invention using the column.

[0013] The present invention also relates to a protein functionally equivalent to the "PRDC" protein. As a method for isolating such a protein, a method for introducing a mutation into an amino acid in the protein is well known to those skilled in the art. That is, for those skilled in the art, for example, "Oligonucleot
ide-directed Dual Amber ”method (Reference: Hashimoto-Goto
h, T., et al. (1995) Gene 152, 271-275., Zoller, MJ and
Smith, M. (1983) Methods in Enzymology 100, 468., Susumu Hirose (1986) Seismic Chemistry Laboratory Lecture 1 "Genetic Research Method II" 10
5., Ausubel, FM, et al. (1987) Current Protocols In M
olecular Biology 1.6.1-1.6.10.), “Gapped duple
x "method (Literature: Kramer, W., et al. (1984) Nucl. Acids Res.
12,9441., Kramer, W. and Frits, HJ (1987) Methods in
Enzymology 154, 350.), "Kunkel" method (literature: Kunkel,
TA (1985) Proc. Natl. Acad. Sci. USA 82, 488., Kunkel, T.
A., et al. (1987) Methods in Enzymology 154, 367.) and “LA PCR in vitro Muta
genesis ”(literature: Ito, W., Ishiguro, H. and Kurosawa,
Y. (1991) Gene 102, 67-70.) And the like, amino acids that do not affect the function of the “PRDC” protein shown in SEQ ID NO: 1 are appropriately substituted by in vitro mutagenesis. Thus, isolating a protein functionally equivalent to the "PRDC" protein is usually a task.
Amino acid mutations may also occur in nature. Thus, the “PRDC” protein (SEQ ID NO: 1)
One or more amino acids in the amino acid sequence in the substitution, having an amino acid sequence is deleted or added,
A protein functionally equivalent to the “PRDC” protein is also included in the protein of the present invention. Here, “functionally equivalent” means that the protein has the same activity of inducing brain nerve differentiation as the “PRDC” protein. The activity of inducing the differentiation of cranial nerves can be examined, for example, as follows. That is, as shown in Example 11 below,
By injecting mRNA of the gene to be analyzed into the Xenopus early embryo and generating the embryo, it is possible to examine whether the gene is involved in the differentiation and formation of brain nervous system tissues.
Alternatively, transgenic animals or mutant organisms in which genes on chromosomes have been disrupted by homologous recombination can be prepared and examined by detecting the effect on the differentiation and formation of brain nervous system tissues. The number of amino acids mutated in a protein functionally equivalent to the “PRDC” protein is not particularly limited as long as it retains a brain nerve differentiation-inducing activity equivalent to the “PRDC” protein. Usually, it is 160 amino acids or less, preferably 50 amino acids or less, more preferably 10 amino acids or less, and still more preferably 1 amino acid or less.
Amino acids. The mutation site may be any site as long as it retains a brain nerve differentiation-inducing activity equivalent to that of the “PRDC” protein.

Other methods for isolating functionally equivalent proteins include hybridization techniques (eg, Sambrook, J., French, EF, Maniatis, T. (ed.)
s): Molecular Cloning, 2nd ed., Cold Spring Harbor La
boratory, 1989, Gubler, U., Hoffman, BJ: Gene, 25: 263-
269, 1983, Hanahan, D .: J. Mol. Biol., 166: 557-580, 1983.
(See, eg, U.S. Pat. That is, those skilled in the art encode the "PRDC" protein.
Based on the DNA (SEQ ID NO: 2) or a part thereof, a DNA highly homologous thereto can be isolated to obtain a protein functionally equivalent to the “PRDC” protein from the DNA. is there. Thus, a protein that is encoded by a DNA that hybridizes with a DNA encoding a “PRDC” protein and that is functionally equivalent to the “PRDC” protein is also included in the protein of the present invention. Here, “functionally equivalent” means that the protein has the same brain nerve differentiation inducing activity as the “PRDC” protein, as described above. Organisms for isolating functionally equivalent proteins include, for example, Xenopus, in addition to mice. The stringency of hybridization for isolating DNA encoding a functionally equivalent protein can be appropriately selected by those skilled in the art. As one specific condition, a hybridization solution (6 × SSC, 5 × Denhardt reagent,
After hybridization using 0.5% SDS, 100 μg / ml denatured salmon sperm DNA, 50% formamide), washing is performed at room temperature in a washing solution having the same composition. The washing temperature is preferably about 42 ° C, more preferably about 65 ° C. In addition, instead of hybridization, it is also possible to use a gene amplification method using a part of DNA (SEQ ID NO: 2) encoding the “PRDC” protein as a primer, for example, a PCR method to isolate.

The present invention also relates to a DNA encoding the protein of the present invention. The DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes cDNA, genomic DNA, chemically synthesized DNA, and the like.
CDNA encoding the protein of the present invention can be prepared by RT-PCR or RACE using mRNA as a template. The genomic DNA encoding the protein of the present invention is a synthetic DNA
Can be obtained by PCR using genomic DNA as a template and genomic DNA as a template, or screening from a genomic DNA library using synthetic DNA as a probe. Also, a chemically synthesized DNA encoding the protein of the present invention.
Can be synthesized by a method such as a solid phase method using a DNA synthesizer.

The DNA of the present invention can be used, for example, for producing a recombinant protein. The host in the host-vector system used for producing the protein of the present invention as a recombinant protein includes Escherichia coli,
Yeast, insect cells, animal cells and the like can be considered, and the vector to be used is specified. As a vector, for example,
Examples of yeast include pHIL-D1 and pHIL-D4. As a method for introducing a vector into a host, biological methods, physical methods, chemical methods, and the like are known to those skilled in the art, and these methods can be used. Examples of the biological method include a method using a viral vector, a method using a specific receptor, and a cell fusion method (HVJ (Sendai virus, polyethylene glycol (PEG), electrical cell fusion method, micronucleus fusion method). (Chromosome transfer)).
Examples of the physical method include a microinjection method, an electroporation method, and a gene particle (genegun). Examples of the chemical method include a calcium phosphate precipitation method, a liposome method, a DEAE dextran method, a protoplast method, an erythrocyte ghost method, an erythrocyte membrane ghost method, and a microcapsule method. As a method for purifying the recombinant protein produced in the host, a known method, for example, a method using an ion exchange column, an affinity column, or the like is used.

The DNA of the present invention can be used for producing transgenic animals and knockout animals.
Further, application to gene therapy is also conceivable. As a vector for expressing the gene of the present invention used for producing a transgenic animal, when specifically expressing in the nervous system, for example, Nestin gene promoter, PRDCcD
A vector containing NA, a polyA addition signal, and a Nestin gene nervous system-specific enhancer in this order is preferably used, and when expressed systemically, it contains, for example, a CMV enhancer promoter, a PRDC cDNA, and a polyA addition signal in this order. Vectors can be suitably used. The transgenic mouse into which the gene of the present invention has been introduced can be produced, for example, by the method described in Example 10. On the other hand, a knockout animal can be prepared by modifying or destroying the DNA of the present invention. The knockout mice of the present invention include both heterozygous knockout mice in which one of the alleles is modified or destroyed and homozygous knockout mice in which both alleles are knocked out.
The knockout mouse of the present invention can be produced, for example, by the method described in Example 9. When the gene of the present invention is used for gene therapy, examples of the vector for gene therapy include, for example, a vector containing a CMV enhancer promoter, a PRDC cDNA, and a polyA addition signal. It is conceivable that the treatment is performed by injecting (eg, nerve cells).

The present invention also relates to an antibody that binds to the protein of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a monoclonal antibody in addition to a polyclonal antibody. A polyclonal antibody can be prepared, for example, by the following method. An equal amount of the recombinant protein produced by an appropriate method is mixed with Freund's complete adjuvant (FCA) to obtain a uniform emulsion. This is injected into about 10 subcutaneous sites of rabbits (New Zealand White, 2500-3000 gr.) Sterilized with 70% alcohol cotton for initial immunization. For the second and subsequent immunizations, Freund's incomplete adjuvant (FIA) is used as an adjuvant. Immunization is performed once every two weeks, and one week after the end of the third immunization, preliminary blood sampling is performed. The obtained blood is centrifuged at 1600 rpm at 4 ° C. to obtain serum. The antibody titer to the protein of the present invention in the serum is measured. When a sufficient increase in the antibody titer is confirmed, the whole blood is collected after a total of 4 to 5 immunizations.
The obtained blood is similarly centrifuged at 4 ° C. and 1600 rpm to obtain serum. This serum is purified using protein A.
After protein A purification, affinity purification of the antiserum is performed using an affinity purification column on which the protein of the present invention is immobilized. In such a process, a rabbit polyclonal antibody is produced. However, the immunized animal is not limited to rabbits, and it is possible to obtain antibodies by immunizing various animals by the same method. Monoclonal antibodies, on the other hand, are based on the method of Koehler and Milstein (Ko
hler, G. and C. Milstein, Nature 256: 495-497 (197
5)) can be created. The antibody thus prepared is used for affinity purification and detection of the protein of the present invention, as well as for diagnosis of diseases (eg, diseases of the nervous system) caused by abnormal expression of the protein of the present invention, and antibody therapy. It can also be applied to such applications. When used for antibody therapy, humanized antibodies or human antibodies are preferred. Humanized antibodies are described, for example, in the literature (Hiroshi Noguchi, Takachika Higashi: Preparation of chimeric antibodies by antibody engineering and its applications, Medical Immunol. 22: 628-638, 1991, Hiroshi Noguchi: Principles of chimeric antibodies and humanized antibodies Clinical Application, History of Medicine 167: 457-462, 1993, Tomoya Nakatani, Hiroshi Noguchi:
Humanization of antibodies, by the method described in Pharmacia 33: 24-28, 1997), and human antibodies are described, for example, in the literature (Chot
hia, C. et al., Nature, 324, 877 (1989), Roguska, ML e
t al., Proc. Natl. Acad. Sci. USA, 91, 969 (1994), Wint
er, G. et al., Annu. Rev. Immunol., 12, 433 (1994), Lonber
g, N. et al., Nature, 368, 856 (1994)).

The present invention also relates to a DNA that specifically hybridizes with a DNA encoding the “PRDC” protein,
It relates to DNA having a chain length of 5 nucleotides. Here, "specifically hybridizes" means that under normal hybridization conditions, preferably stringent hybridization conditions, cross-hybridization with DNA encoding other proteins does not occur significantly. . Such DNA can be used as a probe for detecting and isolating the DNA encoding the “PRDC” protein, and as a primer for amplifying the DNA. Specific primers include, for example, the primers of SEQ ID NOs: 9, 11, and 12.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[0021]

EXAMPLES [Example 1] Preparation and isolation of ES cell line in which genes that control development are trapped In order to efficiently isolate genes, a novel trap vector pIZ was first constructed (Fig. 1). pIZ is pNEB193 (New En
gland Biolabs) by a known method. pI
Z contains the following DNA fragments: E from mouse fgf-4 gene
agI-MscI fragment (containing splicing acceptor site), synthetic D containing termination codons in three different frames
NA, Encephalomyocarditis virus (encepha
lomyocarditis virus) internal ribosomal entry site (IRES)
(S. Jang et al., 1988, J. Virol. 62: 2636-2643), E. coli lacZ gene encoding β-galactosidase used as a reporter, SV40 early mRNA poly (A) addition signal, mouse phosphoglycerate Kinase-
1 (phosphglycerate kinase-1) (CN Adra et al., 1987, Ge
ne 60: 65-74) The neomycin resistance gene of Escherichia coli used as a selection marker. Since IRES creates mRNA with two cistronics,
It is considered that the β-galactosidase protein encoded by lacZ is efficiently expressed when a transcript in which the trapped gene and the trap vector are fused is generated.

The CCE ES cell line used to introduce this trap vector (PL Schwartzberg et al., 1990, Proc.
Natl. Acad. Sci. USA 87: 3210-3214).
emori et al., 1995: Mech. Dev. 51: 265-273). 5 μg of pIZ linearized by PacI digestion was introduced into CCE ES cells (about 1 × 107) by electroporation. After about one week, G418-resistant colonies were selected, and β-galactosidase activity was examined under undifferentiated and differentiated conditions.

Forty-seven G418-resistant colonies were recovered, and the lacZ expression pattern of each cell line was analyzed under undifferentiated and differentiated conditions. When ES cells are differentiated, ES cells are differentiated by culturing in an uncoated plastic dish in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum,
Cell clumps were formed. The cell mass was transferred to a gelatin-coated culture dish and cultured for several days to form an embryoid body. Embryoid bodies were fixed with 0.25% glutaraldehyde and X-gal
Staining was performed. This in vitro differentiation test was mainly used to exclude ES cell lines that trapped a constantly active gene. In vitro differentiation studies showed that 29 cell lines were positive for X-gal staining under undifferentiated conditions, but most of them had weakened signals under differentiated conditions (this resulted in altered reporter gene expression). Rather, it appears to be due to non-specific effects due to reduced cell viability.) On the other hand, one cell line in which lacZ expression was not detected under undifferentiated conditions, but was detected under certain differentiation conditions, was isolated. We named this cell line "B29".

[Example 2] Preparation of chimeric mouse using B29 ES cell line and confirmation of transfer to germ line cells Gene-trapped B29ES in each blastocyst of C57BL / 6 mouse
10-15 cells from cells were injected. Blastocysts were transplanted into pseudopregnant ICR female mice and developed. Chimeric male, C
Crossed with 57BL / 6 female or A / J female. The transfer of the trapped loci to germline cells was determined by
Confirmed by Southern blot analysis of DNA. Southern blot analysis was performed using a probe labeled with 32 P with random primers and was described in the literature (H. Saegusa et al., 1996, Dev. Biol. 174:
55-64). Mice heterozygously carrying the gene trap insertion (GT insertion) (hereinafter referred to as "heterozygous mice" or simply "hetero") were normal. These were crossed to produce mice homozygous for the GT insertion (hereafter referred to as "homo mice" or simply "homo"). Homozygous mice were viable and showed no obvious abnormalities.

[Example 3] Expression patterns of lacZ and TAG-1 in gene-trapped embryos To examine the expression pattern of the trapped genes and TAG-1, a marker gene for commissure nerves, during development, heterozygous embryos were examined. X-gal staining and immunohistochemical staining of TAG-1 were performed using the same.

In the usual case, embryos are fixed in a fixative (0.2% glutaraldehyde, 1.2% formaldehyde, and 0.02% NP-40).
Was dissolved in PBS) for several hours and then washed with PBS. Then, X-gal solution (8.4 mM KCl, 1 mM MgCl2, 3
mM K4Fe (CN) 6, 3 mM K3Fe (CN) 6, 0.05% NP-40, and 0.05% 5-bromo-4-chloro-3-indolyl-β-D-galactop
yranoside in 84 mM phosphate buffer pH 7.5).

Double staining of X-gal and anti-TAG-1 antibodies was performed as follows: GT insertion 11.5 or 12.5 days (E11.5 or E12.5) post-coitum. Embryos having a homozygote are fixed with a 4% glutaraldehyde solution in PBS at 4 ° C. for 1 hour, developed with an X-gal solution at room temperature overnight, and washed with PBS.
The cells were fixed again with a 4% glutaraldehyde solution in PBS at 4 ° C. for 1 hour, and finally washed again with PBS. Embryos stained with X-gal were cut into several parts along the anterior-posterior axis, and immunohistochemical staining was performed with an anti-TAG-1 antibody (R. Shirasaki et al., 1996,
Neuron 17: 1079-1088). Immunohistochemical staining is essentially performed by Davis (CA Davis, 1993, In: Guide to Techniques
in Mouse Development (Eds PM Wassarman & ML
DePamphilis), pp. 502-516. Academic Press, San Di
ego, CA.) (3,3′-diamizobenzidine was used as a substrate for horseradish peroxidase conjugated to a secondary antibody). The double-stained embryos were re-fixed with Bouin's solution to prepare paraffin sections.

As a result, X-gal staining was first detected in the central nervous system and a part of the digestive tract in the posterior midbrain of E10.5. Central nervous system signals were distinct up to E12.5 and decreased at E13.5 (FIGS. 2A-D). At E14.5 and E15.5, X-gal staining was detected almost systemically (FIGS. 2E, F) due to lacZ expression in the dermis.

In X-gal staining on transverse sections of the tail of the spinal cord of heterozygous E12.5 embryos, the signal appeared to be localized to commissure nerves (FIG. 3A). Experiments using an anti-TAG-1 monoclonal antibody as a molecular marker for commissural nerves confirmed this possibility. First, homozygous E11.5 and E12.5 were stained with X-gal, and then immunostained with anti-TAG-1 antibody. (There was no difference in the X-gal staining pattern between heterozygous and homozygous embryos. , Only homozygous embryos with stronger signals were used). E11.5 lumbar spinal cord (lumberspin
al cord), the two signals overlapped. That is, X-gal staining is localized in the dorsal nerve axon, and TAG-1 immunostaining is also localized in the dorsal nerve axon where the cell body is located on the farthest back. (Figure 3B). this is
This suggests that the cells expressing lacZ are commissural nerves. However, at E12.5, the signals from the two staining methods did not overlap. Commissural nerve axons that express TAG-1
Lac-Z expressing cells were localized near the center of the spinal cord with respect to the dorsal-ventral axis, as observed from the dorsal side of the spinal cord to the floor plate (FIG. 3C). X-gal staining was also detected on the floor plate and appears to be derived from lacZ-expressing nerve axons (axons extending from lacZ-expressing cell bodies were observed in embryo double staining, With paraffin sections, the signal was weak and difficult to detect).
From this, it was inferred that cells expressing lacZ in E12.5 were commissure nerves not expressing TAG-1. Differences in X-gal staining patterns in the lumbar and tail spinal cord of E12.5 (Figures 3A and C) may reflect differences in the stage of neurogenesis along the anterior-posterior axis Conceivable. Axons expressing lacZ and not immunostained with TAG-1 are also observed on the ventral side of the E11.5 lumbar spinal cord (Fig. 3
B). This suggests that there is a group (or groups) of other nerves that express the reporter gene.

[Example 4] Cloning of 5 'of trapped endogenous gene Using 5'-RACE method, cloning of cDNA derived from 5' side of vector insertion site in predicted fusion transcript Was done. 5'-AmpliFINDER RACE Kit (Clontech)
Used according to manufacturer's instructions. The primer sequences used are as follows. "APS" (SEQ ID NO: 3 / 5'-C
CTCTGAAGGTTCCAGAATCGATAG-3 '), "IR1" (SEQ ID NO:
4 / 5'-TGCCAAAAGACGGCAATATGGTGGA-3 '), "IR2" (SEQ ID NO: 5 / 5'-TCGTCAAGAAGACAGGGCCAGGTTT-3'), "IR
4 "(SEQ ID NO: 6 / 5'-CATATAGACAAACGCACACCG-3 '),
"SA1" (SEQ ID NO: 7 / 5'-AATTCCGCACCGAGAGACCA-
3 '). AGPC method (P. Cho)
mczynski & N. Sacchi, 1987, Anal.Biochem. 115: 41
9-423), and poly (A) + RNA was concentrated using oligo (dT) latex (Oligotex-dT30 <Super>, manufactured by Nippon Roche). The first strand of cDNA was synthesized using the IR2 primer corresponding to the IRES sequence. After ligation of the anchor with T4 RNA ligase, IR1 located upstream of IR2 of the APS and IRES sequences corresponding to the anchor sequence
PCR was performed using the primer pair (94 ° C. for 30 seconds, 60 ° C.
5 seconds, 72 ° C, 2 minutes, 35 cycles). Nested PCR (nested PC
R) was performed using IR4 and SA1 as antisense primers located upstream of IR1 and APS as sense primers (94 ° C for 30 seconds, 64 ° C for 1 minute, 72 ° C for 2 minutes, 35 cycles).
An approximately 250 bp fragment (B2
9U) was amplified. This fragment was transferred to the pGEM-T vector (Promega
And the sequence was performed. Sequence is SEQUENASE version 2.0.7-deaza-dGTP Kit
(USBiochemicals) or ABI Prism 310 Genetic
Analyzer (Perkin-Elmer) was used.

Example 5 Molecular Cloning of cDNA Derived from Trapped Endogenous Gene First, a gene expressed in the mouse central nervous system is included.
A cDNA library was prepared as follows.

The E11.5 embryo (strain C57BL / 6) was roughly cut into dorso-ventral regions, and the dorsal side (including the central nervous system) was defined as a central nervous system fraction. The AGPC method (P. Chomcz
ynski & N. Sacchi, 1987, Anal. Biochem. 115: 419-4
In 23), total RNA was isolated. Then, using an oligo dT cellulose column (Type 7, manufactured by Pharmacia), poly (A)
+ RNA was isolated. Using this poly (A) + RNA, two plasmid cDNA libraries were prepared. One is a random primer (RP) library, which uses random hexamers as primers for first-strand synthesis, and the other is a linker primer (LP) library, which uses oligo (dT) and
A linker primer having an XhoI recognition site (SEQ ID NO: 8 /
5'-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTT
TTT-3 '(Stratagene) was used. In both cases,
First-strand synthesis using cDNA Synthesis kit (Stratagene) in the presence of 5-methyl dCTP, first 2.5 μg of poly (A) + RNA
Performed using In the preparation of the RP library, the cDNA to be inserted was ligated with an EcoRI adapter, and ligated with EcoRI-digested pBluescript SK (-) (Stratagene). To construct an LP library, insert the cDNA
After ligation of the EcoRI adapter to XhoI, the resulting cDNA was digested with XhoI, and the resulting cDNA was double digested with EcoRI and XhoI.
It was ligated to Bluescript SK (-) (cDNA was inserted in the direction in which the 5 'end becomes an EcoRI site uniquely). Each library was electroporated using E. coli XL-1 Blue MRA (P
2) (Stratagene) and cultured overnight. Some of the cultured Escherichia coli was -15% in the presence of 7% dimethyl sulfoxide.
Stored frozen at 0 ° C. A plasmid was prepared from the remaining culture solution, and the amplified plasmid library was used as a template for PCR. For the purpose of searching for the “PRDC” cDNA, Escherichia coli into which the plasmid library was introduced was transplanted to an LB plate, and colony hybridization was performed using the B29U fragment obtained in Example 4 as a probe (about 5 × 10 5
Colonies). As a result, two clones with overlapping regions from the RP library (pRP4-1 and pRP8-6)
Was isolated. pRP8-6 retained the longer insert (about 850 bp) and completely contained the insert for pRP4-1 (about 400 bp). Northern blot analysis was performed using these 32P-labeled probes with random primers according to the literature (H. Saegusa et al., 1).
996, Dev. Biol. 174: 55-64). As a result, the size of the mRNA of this trapped gene was shown to be about 3.7 kb (FIG. 6), and the obtained clone was presumed to be a partial sequence.

Example 6 Cloning of 3 ′ of Trapped Endogenous Gene In order to clone the entire gene, LA-PCR (manufactured by Takara) was further performed using an LP cDNA library as a template. The reaction was performed at 30 cycles of 94 ° C. for 30 seconds, 64 ° C. for 1 minute, and 72 ° C. for 3 minutes. As a primer pair, a PRDC sequence-specific primer “U4” (SEQ ID NO: 9 / 5′-GCTGTCTCCAGCTCCTTCCA
CATCTGCAGG-3 ') and vector sequence-specific primer "T7" (SEQ ID NO: 10 / 5'-GTAATACGACTCACTATAGGGC-3')
Was used.

The PCR product was blunt-ended with T4 DNA polymerase and degraded with BamHI (the BamHI site appeared to be one in the trapped cDNA gene). The obtained DNA is Hin
Ligation was performed on pUC19 which was double digested with cII and BamHI. The trapped cDNA gene was screened by colony hybridization using pRP8-6 cDNA (see Example 5) as a probe. As a result, BamU of B29U
PPB1-3-1 containing 3.6 kb downstream from the I site was obtained.

Example 7 Analysis of Trapped Endogenous Gene Sequence When B29U, pRP8-6 and pPB1-3-1 cDNAs were combined, almost the entire cDNA of the trapped gene was covered. By sequence analysis, these clones had a total length of 3764 bp (FIG. 4 and SEQ ID NO: 2),
Turned out to be about the same size. In order to confirm the sequence of the cDNA, PCR was performed using several primer pairs specific to the sequence of pRP8-6 and / or pPB1-3-1 and the LP library plasmid as a template, and the obtained DNA was obtained.
The fragments were sequenced.

According to the sequence analysis, the longest open reading frame was from nt 249 to nt 755, and the trapped gene encoded a novel protein consisting of 168 amino acids (FIG. 4 and SEQ ID NO: 1 and
2). Since the predicted protein has a hydrophobic region at the amino terminus, it appears to be a secreted protein or a membrane protein. However, since this gene product does not have a region presumed to be a transmembrane region, it is highly likely that the gene product is a secretory protein. Structurally, there were no known motifs except at two potential N-linked glycosylation sites (Figure 4). 3 '
The long untranslated region flanked by AUUUA is AT-rich and is known to be involved in mRNA destabilization (C.-YA Chen & A.-B.Shyu, 1995 , T
rends Biochem. Sci. 20: 465-470).

According to a database search using the BLAST method and the FASTA method, this trap gene product was found to be rat Dr.
m (LZ Topol et al., 1997, Mol. Cell. Biol. 17: 4801-4
810) and moderate sequence homology to mammalian DA
N (T. Ozaki & S. Sakiyama, 1993, Proc. Natl. Acad. S
ci. USA 90: 2593-2597; H. Enomoto et al., 1994, Oncigen.
e 9: 2785-2791; T. Ozaki et al., 1996, Jpn. J. Cancer R
es. 87: 58-61), mouse Cerberus-like (JA Belo
68, 45-57; C. Biben et al., 1998, Mech.
Dev. Biol. 194: 135-151), and Cerberus (T. Bouwmeester) expressed in the region of the formed body of Xenopus embryos.
Et al., 1996, Nature 382: 595-601). Among them, FIG. 5A shows the amino acid homology of “PRDC”, DAN and Cerberus. The region of homology was limited and spanned about 90 amino acid residues. In the homologous region, the proportion of matching of the amino acid residues with the trapped gene was approximately 69% (79/113) that of rat Drm, and that of mouse Drm.
AN is about 37% (33/90), and mouse Cerberus-like is about 25
% (16/63), and about 29% (26/89) with Xenopus Cerberus. FIG. 5B summarizes the amino acid homology of the homologous regions of the gene products of the trap gene, mouse DAN, and Xenopus cerberus in the present invention. Since the cysteine residues in the homologous regions are well conserved, these three gene products are likely to have similar tertiary structures. Based on such agreement, the present inventors have designated this trap gene as "PRDC" (protein relat
ed to DAN and cerberus).

Example 8 Comparison of Expression Pattern of Endogenous “PRDC” with Expression of TAG-1 First, a mouse TAG-1 cDNA fragment was subjected to PCR using the E11.5 central nervous system plasmid cDNA library as a template. Isolated. The reaction was performed at 35 cycles of 94 ° C. for 30 seconds, 64 ° C. for 1 minute, and 72 ° C. for 2 minutes. Completely sequence-matched portions of rat and human TAG-1 were used for the primer pair. That is, "TAG1-F
1 "(SEQ ID NO: 11 / 5'-ATCAAGTGGCGCAAAGTGGA-3 '),
"TAG1-R1" (SEQ ID NO: 12 / 5'-TGCCCATGAAGTTCTCAGCA
-3 ') was used as a primer pair. The fragment (about 670 bp) amplified by PCR was blunt-ended with T4 DNA polymerase and cloned into the SmaI site of pBluescript SK (-) (Stratagene). Since the sequence of the amplified fragment showed high homology with rat and human TAG-1, it was estimated to be a part of mouse TAG-1 cDNA. This plasmid was used as a template for RNA probe synthesis.

The in situ hybridization was performed as follows. Embryos were dissected and removed in PBS and fixed with 4% glutaraldehyde solution in PBS on ice for 5-6 hours. Embryos were dehydrated in an ethanol / butanol series and embedded in paraffin. The procedure for in situ hybridization of paraffin sections is described in Wilkinson and Nieto (DG Wilkinson & MA Nieto, 1993, In: G
uide to Techniques in Mouse Development (Eds PM
Wassarman & ML DePamphilis), pp. 368-370.
emic Press, San Diego, CA.). "PRD
Digoxigenin (DIG) labeled RN for detection of `` C '' mRNA
The A probe was used (corresponding to nucleotides 166 to 847 in FIG. 4). The TAG-1 probe was similarly synthesized using DIG-labeled UTP.

As a sample, wild-type mouse embryo (C57BL / 6 strain)
Embryos of various stages were used. The expression of “PRDC” is E1
First detected in 0.5 spinal cord. From E10.5 to E11.5, `` P
"RDC" signals were observed in a group of nerves located at the dorsal edge of the spinal cord (FIG. 3D). At the same stage, TAG-1 is expressed in a wider range, including "PRDC" expressing cells,
In addition, expression was found in the ventral cells and dorsal root ganglia, which are considered to be motor nerves (FIG. 3E). These TAG-1 expression patterns were consistent with those observed in the spinal cord of rat embryos (A. Furley et al., 1990, Cell 61: 157-170). TAG
Among the -1 expressing cells, the dorsal side of the spinal cord is thought to be the commissure nerves, so it was found that "PRDC" was also expressed in a group of commissure nerves. The expression level of `` PRDC '' is E11.
Decreased between 5 and E12.5. In E12.5, expression of "PRDC" was detected only in the commissure nerves of the spinal tail. E13.5
Thereafter, the signal of “PRDC” was not detected.

Example 9 Preparation of “PRDC” Knockout Mouse Poly (A) + RNA prepared from a wild-type, heterozygous, or homozygous E11.5 embryo was analyzed by Northern blot, and the insertion of the trap vector was determined to be “PRDC”. The effect on gene expression was examined (FIG. 6). A 500 b fragment containing the 5 ′ untranslated region and a part of the coding region of “PRDC” was used as a probe (this probe overlaps only about 90 b with B29U (upstream sequence of the vector insertion site). After washing, the probe is unlikely to detect fusion transcripts from the trapped loci). A 3.7 kb band was observed from the wild-type embryo sample. Heteroembryonic samples had the same size but lower signal intensity. In homozygous embryo samples, a thin band of the same size was observed. This is a small amount of wild-type “PR” from homozygous embryos.
DC "suggests that mRNA is being produced. According to densitometric analysis, mRNA levels in homozygous embryos
About 10% of that of the wild-type embryo. Furthermore, RT-PCR (reverse transcription-PCR) was performed on RNA collected from homozygous embryos, and it was confirmed that wild-type “PRDC” mRNA was generated from homozygous embryos. This suggests that the trapped "PRDC" locus is not null, but a hypomorphic allele. Therefore, using a targeting vector created so that the region encoding the “PRDC” gene is completely deleted,
DC "knockout (KO) mice were generated as follows. First, the targeting vector was introduced into ES cells (129 / Sv strain mouse) by electroporation. ES cells having the desired mutation were identified by PCR and Southern blotting. The mutant ES cells were identified as C57BL / 6 Strain mouse 3.5
The embryos were injected into the embryo cavities, which were then implanted into foster parents. Among the born mice, mice judged to be chimeric mice based on coat color (those that have some brown coat color) were replaced with male or female
The mice were bred with C57BL / 6 strain mice. Among the newborn pups, the pups with brown hair were analyzed by PCR to determine whether the PRDC gene was mutated. As a result, PRD
Hetero mutant mice in which the C gene was mutated (hetero KO mice) were obtained. The heterozygous mutant mice were crossed to obtain a homozygous mutant mouse (homoKO mouse). Fabricated hetero
KO mice were apparently normal. Homo KO mice were born normally and developed abnormally for at least 2 weeks. Thus, "PRDC" did not appear to be essential for mouse fetal development.

Example 10 Preparation of Transgenic Mice Expressing Ectopic Expression of “PRDC” In order to clarify the function of “PRDC” in individuals, “PRD” was used.
Transgenic (T) that ectopically expresses “PRDC” throughout the fetal central nervous system by constructing a vector in which the “C” gene is linked downstream of the nestin nervous system-specific enhancer.
g) A mouse was prepared. Specifically, a vector containing a Nestin promoter, a PRDC cDNA, a polyA addition signal, and a Nestin nervous system-specific enhancer in this order was injected into a mouse fertilized egg, and this was transplanted to a foster parent. Whether or not the born mouse had the introduced DNA was analyzed by PCR to obtain a target Tg mouse.

The Tg mouse thus produced grew normally in appearance, and no abnormality was observed in fertility. When male wild-type mice and female Tg mice or male Tg mice and female wild-type mice were crossed, wild-type and Tg pups were obtained from both. Offspring born and raised by Tg mice grew slower than offspring born and raised by female wild-type mice. When female pups of wild-type mice were raised by Tg mice, growth was slow, and when pups of female Tg mice were raised by wild-type mice, growth was normal. Since female Tg mice were normal in their lactation behavior, these results indicate that ectopic expression of `` PRDCs '' during fetal life could lead to any abnormalities related to milk production, including but not limited to abnormal development of the mammary gland Was suggested to occur.

Example 11 Assay of Secondary Axis Inducing Activity of "PRDC" in Xenopus Embryos "PRDC" is known to form a secondary embryo head and body axis in Xenopus embryos. There is low homology with cerberus on the amino acid sequence of the translation product. Therefore,
The effect of the "PRDC" gene on Xenopus embryo development was analyzed. "PRDC" mRNA, or lacZ mRNA as a negative control, was injected into various sites of Xenopus 1-16 cell stage embryos and the effect was examined (FIG. 7). As a result, no clear change was observed in the embryos injected with lacZ mRNA (FIG. 7B), but in the embryos injected with “PRDC” mRNA, although the frequency was different depending on the injection site, secondary changes were observed. The formation of a large head and body axis was observed (FIG. 7A).
LacZ mRNA was injected together with “PRDC” mRNA, and β-galactosidase activity was detected by X-gal staining. As a result, only the secondary formed head was stained (FIG. 8). Therefore, "PRDC" is cerb in Xenopus embryos.
It was shown to have secondary axis inducing activity similar to erus.

[0045]

Industrial Applicability According to the present invention, a novel protein involved in the differentiation of brain nerve tissue cells, DN encoding the protein
A, a vector into which the DNA has been inserted, a transformant carrying the DNA, and a method for producing a recombinant protein using the transformant. Further, the present invention provides antibodies and oligonucleotides used for detection, isolation, and the like of the protein and the DNA. Further, the present invention provides a transgenic mouse into which the DNA has been introduced and a knockout mouse in which the DNA has been modified or destroyed. Since the protein of the present invention and its gene are considered to be involved in the induction of differentiation of the cerebral nervous system, they are useful for elucidating the formation mechanism of cerebral nervous system tissues. Further, the proteins and genes according to the present invention and the compounds that regulate the functions of the proteins are expected to be used for prevention and treatment of diseases of the cerebral nervous system.

[0046]

[Sequence List] (1) Name or name of applicant: Meiji Dairies Co., Ltd. Kanagawa Academy of Science and Technology (2) Title of the invention: Novel protein involved in differentiation of brain nerve tissue cells (3) Reference number: M1-002 (4) Application number: (5) Filing date: (6) Number of sequences: 12 Sequence number: 1 Sequence length: 168 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Phe Trp Lys Leu Ser Leu Thr Leu Leu Leu Val Ala Val 1 5 10 Leu Val Lys Val Ala Glu Thr Arg Lys Asn Arg Pro Ala Gly Ala Ile 15 20 25 30 Pro Ser Pro Tyr Lys Asp Gly Ser Ser Asn Asn Ser Glu Arg Trp His 35 40 45 His Gln Ile Lys Glu Val Leu Ala Ser Ser Gln Glu Ala Leu Val Val 50 55 60 Thr Glu Arg Lys Tyr Leu Lys Ser Asp Trp Cys Lys Thr Gln Pro Leu 65 70 75 Arg Gln Thr Val Ser Glu Glu Gly Cys Arg Ser Arg Thr Ile Leu Asn 80 85 90 Arg Phe Cys Tyr Gly Gln Cys Asn Ser Phe Tyr Ile Pro Arg His Val 95 100 105 110 Lys Lys Glu Glu Asp Ser Phe Gln Ser Cys Ala Phe Cys Lys Pro Gln 115 120 125 Arg Val Thr Ser Val Ile Val Glu Leu Glu Cys Pro Gly Leu Asp Pro 130 135 140 Pro Phe Arg Ile Lys Lys Ile Gln Lys Val Lys His Cys Arg Cys Met 145 150 155 Ser Val Asn Leu Ser Asp Ser Asp Lys Gln 160 165 SEQ ID NO: 2 Sequence length: 3764 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA sequence characteristics Characteristic symbol: CDS Location: 248 .. 752 Method for determining characteristics: E sequence GTGCTGTCTC CAGCTCCTTC CACATCTGCA GGCGCGGCTG GGACGCGCTC CGCCCTCTGT 60 GCGCTCCACT GGGCTCCGCT CGCTCCGCAC ACCCTCGCGCCT CTTCCCAGGCGACTCTTCCCGAGCCGCT AGACACACAA GGGAACCGAG GAGGAGGCTT CCATCTCGTC 240 ATTGCAGG ATG TTC TGG AAG CTC TCG CTG ACC TTG CTC CTG GTG GCT GTG 290 Met Phe Trp Lys Leu Ser Leu Thr Leu Leu Leu Val Ala Val 1 5 10 CTG GTA AAG GTA GCT AGA CAG CAGC AGAGG GCG GGC GCC ATC 338 Leu Val Lys Val Ala Glu Thr Arg Lys Asn Arg Pro Ala Gly Ala Ile 15 20 25 30 CCC TCG CCT TAC AAG GAT GGT AGC AGC AAC AAC TCA GAG AGG TGG CAT 386 Pro Ser Pro Tyr Lys Asp Gly Ser Ser Asn Asn Ser Glu Arg Trp His 35 40 45 CAC CAG ATC AAG GAG GTG CTG GCC TCC AGC CAG GAG GCC CTG GTA GTC 434 His Gln Ile Lys Glu Val Leu Ala Ser Ser Gln Glu Ala Leu Val Val 50 55 60 ACC GAG CGC AAG TAC CTC AAG AGT GAC TGG TGC AAG ACG CAG CCT CTG 482 Thr Glu Arg Lys Tyr Leu Lys Ser Asp Trp Cys Lys Thr Gln Pro Leu 65 70 75 CGG CAG ACA GTG AGC GAG GAG GGT TGC CGC AGC CGC ACC ATC CTC AAC 530 Arg Gln Thr Val Ser Glu Glu Gly Cys Arg Ser Arg Thr Ile Leu Asn 80 85 90 CGC TTC TGC TAC GGC CAG TGC AAC TCC TTC TAC ATC CCG CGA CAC GTG 578 Arg Phe Cys Tyr Gly Gln Cys Asn Ser Phe Tyr Ile Pro Arg His Val 95 100 105 110 AAG AAG GAG GAG GAC TCC TTC CAA TCC TGC GCT TTC TGC AAG CCC CAG 626 Lys Lys Glu Glu Asp Ser Phe Gln Ser Cys Ala Phe Cys Lys Pro Gln 115 120 125 CGT GTC ACC TCT GTC ATC GTA GAG CTC GAA TGC CCG GGT CTC GAC CCA 674 Arg Val Thr Ser Val Ile Val Glu Leu Glu Cys Pro Gly Leu Asp Pro 130 135 140 CCT TTC CGA ATC AAG AAA ATC CAG AAG GTG AAG CAT TGC CGG TGC ATG 722 Pro Phe Arg Ile Lys Lys Ile Gln Lys Val Lys His Cys arg Cys Met 145 150 155 TCA GTG AAC CTG AGT GAC TCC GAC AAG CAG TGAACTCATG GGGCAGGATG 772 Ser Val Asn Leu Ser Asp Ser Asp Lys Gln 160 165 CAGCTCAGCC TTGAGCACTC CTACCCCTGG GTTGCGCCAC CGCCACTTCT GTTGAGCTCA 832 CTCACACTCT GCCCGGTGTC CCAGTCAGCA GCAAATGTGT CTCTTAGGGC CAACAGTGTC 892 CCTGTCACAA ATGAGGACCA CAAGTGAAGC TTTGTCAACT GTCCCAGATC CACCCCAGGC 952 TACACTTGTG TCTCTAAATC CACTTTGTGG ATGGGACACA GAAGATGTTG GAAAACTCCA 1012 CTCCCAGAAA CTCGACAAGA TTTTTCAGGA GATGCACTGT CATCTGGGAT TGACGACTCC 1072 ACCATGTGGA AAAGCCAGTC TTTCCCTGGC CACATCCTAC ACTCCAGCTT GAGCCTTCCT 1132 CTGGTGAAGG ATGCACGACT CTTCTGTCGC AAACTTTTGT TGGTCACCGG ACCTCTGTGG 1192 TGTGAACATT CTTTCTTCTG ACCTGGGAGT TGCCCAGCTC TGCCGCTGCT GTGATGCTGA 1252 TGTGGGCAGC ATCTATAAAG GAGATGCTCA CCCGCAAGGG GTTCCAGAAC AAATCTGGTA 1312 CCCACGAGGA CAAGCCAAAC CAAG TGGATC AGGAAATGGA CTTCCCTAGG AACTGTACTG 1372 AGCAGGCAAG ACTCTCCACA CCTCTGGACA CCACCACCTC TTTATTTCCT TCTCATAGCC 1432 CCTATCTGTA GCTCAAGATA AACTCTGGTG TGCATGCTAT TCCTGCTGCT GTGTTAACAC 1492 TCACAAGGTC TCTGTATTAG TGTCAGTCTT ATAATTGACC TATCATGTTA AAAACAGTTA 1552 AAAGTATTAT CAACATATTC ATATATCAAG TCACGTGTTT GGACTCTAGC TTAGCATCAC 1612 TGTTCTGAAT AGGGAGTAGA GCATCGAGTG ATCTGTGGAA AAGTATTTTA CAGAGGAAAA 1672 ACTGCCAGGT CAGCAATTCA CTCCCCAACC CACCCCCCAC CCCCCCACCC CCGCTTGCTC 1732 ACACACACTC TCTCTTTCCA GATTGAGCAG GGAGATGGGA CACATCATGA TTTTACTTTT 1792 AATATGACAT CACAGGAGAG ACTAAAGCAT TGTGGCATAG TGTACAAACC GGATTGGGAC 1852 AGAGGTGTGT ACAGAGGAGT CACTAGGAAG CTGTAAAGAT TAAAAGGCAC GTGTGAGAGG 1912 AGACCTGGTA CTGTTTTCAC TGCCATTACA CTAAGTAAAA AAATATTTTC TATTAAAAGA 1972 TATGTAAGAA TATATGGTAT TGAGACATTT TGTGACACAG GCAGAAATAT AGAGCCAATA 2032 TAGAGGGTAG TAATGTAGGT AAACTGTCAC TCAGAGGATA GCTTCCTGTA CAAAGAATAG 2092 ACTCCTAAAA TACCAAGAAA CATTTCAGTC ATCTTCAGTT CTGCTGATGA GTTGTTGGAG 2152 TCTCTGTTGT GTGGTCTTTT GATGCGAGGA CTTTGAAATA ATTTATTTTT CCTTCAAAAT 2212 TACACTCAAG TTCAATCTCT TTGTGCAATT GTTCTAGGGA GGCCTCCCTG AGAATGGCAC 2272 GTATGCAATC TGACAAATGT GAATGAGAAA CCTTGGTGGG AATCCGAATG TGCTGAAATC 2332 CTTTGGTTGA TAATTTCCCA GTCCATGCTC TTGCCAAGAT GGGTGTCCTA GTATGTGAGG 2392 GAGACTATCC ATAAGCTGCT CTGAGACTCA GGCTTGTGGA TGCCCAGCAG TGATGCTGGC 2452 TCTCCAGTCT GCCACCTGGA GGAGGGTTGG GATGAGCTGG GGAGGGTATG CCTTGGACTC 2512 TTTTGCCTGT ATTGCAATGT AAGTTTTCTA GTTTCATCAC TTCTGCTACA CAGCAACCAT 2572 GAGTAGAGAC TATGTGGTTT TGAAGGGGAT ATTAGCTTCA GAAATGAGAG TGCACAGCCT 2632 GGAATCAGCA GAGAGAAACA AGGGGGAAAT GGAAAAATCA CAGAGGGCTG GAAACCCCAG 2692 GGATAAAGGC AAAAAGGGAA GAGGGTGCCA AAGGTCAGAG ATACAATTTT CACTTTTAGC 2752 AGAAAATCTC GAGGTAGAGC CAAAGCAGAA CATGCAGTAG AAAAGCTGTG AAAGAGATAT 2812 GAGTGTCCTT AATGGAGGAT GGTGACATGT AACCGGAGGG TGAGGAGAAG AGTCTCACAC 2872 CCAGCTGCGA AGCCAGCCCA TGTCATTCTA TTAATGGGAC CCACACATCA CCAGGAAAGG 2932 TTAAGCTAGG GACCCACAAT GTATGTATGT GAGCATTCTT TTTGATCCTA ATGGTGGGTG 2992 TACCCATGAA GGCTATATGA TATGCTGTAG TCTGA GGCTA CTTCTTGGCT TATTGTATGT 3052 ATAAAGTTTA CCTTCGAATG GCTCAAATTG TATTTAAATT GTGTCTTGTT TATAAATGAA 3112 GAAAAGCTAT AAGTATAATG TAATTATTTT ATAGGTATAC TATTAAGTTA TAGAACAAAT 3172 AAAGATACTC TAGGATTCTA TGTGATCCTG GCATCATGTG CCATAAAAGC TGAATTCTGC 3232 ACTTGTGTCC TTGTGTACTC AGAGTGCATT TATCCAGCCT CTGAGACTTC ACCCACGGTC 3292 CGTATCATAA ACGTTCCTTC GGGGGGCTTG GGATCTTCTA GGAGAATTAG AAACAGGCAT 3352 CAAACAAGAA GAATTGGTGG GATGGCTCTC ACCTCCAGTC TATGGGAACT TCAAGCCTTT 3412 ATTGAAACAC CCACCAGCTG AATACAGAAT GTACTCAGAG TGTTCTTGGA ATAAGAAATA 3472 TTGAATGAGG AAGCTTTGAG AGACAGATGC AAAGAGAAGT CCACTGTAAG TCTCTTCTTC 3532 TCTCCTGTAG CCAGTGCACC ACACTTGTGT CTTACAGAAG CCAGAGATTG TATTGTTTTG 3592 TTCTATTTTA TGTTTACTTT CCACGGGTTT CAACATTTCT TAATTTAAAC ACATCTCAGC 3652 CTCACAGTGT TGTCATCTTC CTGCAAACTG AGTTATCTGC CATTTAAGAC ATGCAATGAT 3712 CAATAAAAGA AATAAAAACC TTGACAAAAC TGCAAAAAAA AAAAAAAAAA AA 3764 SEQ ID NO: 3 sequence length of 25 sequences Type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence species : Other nucleic acid Synthetic DNA sequence CCTCTGAAGG TTCCAGAATC GATAG 25 SEQ ID NO: 4 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TGCCAAAAGA CGGCAATATG GTGGA 25 SEQ ID NO: 5 Sequence length: 25 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TCGTCAAGAA GACAGGGCCA GGTTT 25 SEQ ID NO: 6 Sequence length Length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CATATAGACA AACGCACACC G 21 SEQ ID NO: 7 Sequence length: 20 Sequence type: Nucleic acid chain Number: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA sequence AATTCCGCAC CGAGAGACCA 20 SEQ ID NO: 8 Sequence length: 50 Sequence type: Nucleic acid Number of strands: single-stranded Topology: linear Type Sequence type: Other nucleic acid Synthetic DNA sequence GAGAGAGAGA GAGAGAGAGA ACTAGTCT CG AGTTTTTTTT TTTTTTTTTT 50 SEQ ID NO: 9 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA sequence GCTGTCTCCA GCTCCTTCCA CATCTGCAGG 30 SEQ ID NO: 10 Sequence Length: 22 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GTAATACGAC TCACTATAGG GC 22 SEQ ID NO: 11 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATCAAGTGGC GCAAAGTGGA 20 SEQ ID NO: 12 Sequence length: 20 Sequence type: Nucleic acid Strand number: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TGCCCATGAA GTTCTCAGCA 20

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a pIZ trap vector. SA: splicing acceptor, Ter: termination codon in all three reading frames, IRES: internal ribosomal entry site of encephalomyocarditis virus (encephalomyocarditis virus)
site), lacZ: Escherichia coli lacZ gene encoding β-galactosidase, poly (A): SV40 poly (A) addition signal, Ppgk: phosphoglycerate kinase-1 (phosphgl
neo: promoter of ycerate kinase-1), neomycin resistance gene of Escherichia coli. PNEB19 as the underlying vector
3 was used.

Fig. 2 LacZ of embryo with heterozygous trapped loci
It is a figure showing the change in the development stage of expression. Embryos with a heterologous GT insertion were stained with X-gal (blue signal). (A) E1
0.5, (B) E11.5, (C) E12.5, (D) E13.5, (E) E14.
5, (F) E15.5. The bar of panel (A) (B) (C) is 1mm,
The bars on panels (D), (E) and (F) represent 2 mm.

FIG. 3 is an in situ hybridization showing expression of “PRDC” and TAG-1. (A) Cross section of the spinal cord tail of a sample obtained by X-gal staining of an E12.5 embryo having a heterologous GT insertion. (B) or (C) is a homozygous embryo E11.
5 is a transverse section of the lumbar spinal cord of a sample double-stained with X-gal and anti-TAG-1 antibody for 5 (B) or E12.5 (C). In (B), the immunostaining signal (brown) is the X-gal signal (blue)
However, in (C), the cells expressing lacZ and the associated nerve axon that reacted with the anti-TAG-1 antibody are not positionally identical. (D) or (E) In situ hybridization with digoxigenin-labeled "PRDC" (D) or TAG-1 (E) riboprobes using adjacent paraffin sections of the lumbar spinal cord of wild-type E11.5 embryos. . The bar is
Represents 100 μm.

FIG. 4 is a diagram showing the nucleotide sequence and predicted amino acid sequence of “PRDC” cDNA. The poly (A) addition signal is underlined. ATTTA repeats that are associated with mRNA instability are shown in bold. Arrowhead (nucleotide 246/24
7) represents a vector insertion site. The expected signal peptide sequence is boxed. The signal peptide cleavage site was determined by von Heijne (G. von Heijne, 1986, Nucleic Acids Re
s. 14: 4683-4690). Expected N-glycosylation sites are indicated by shaded letters.

FIG. 5 shows the homology of amino acid sequences of “PRDC”, DAN, and Cerberus. (A) Xenopus C
erberus (top), mouse DAN (bottom), and "PRDC"
It is a figure which shows the homology region of (middle). Those that correspond to the amino acid residues of "PRDC" are boxed. Conserved cysteine residues are shaded. Xenopus Frog Cerb
The sequence of erus is described in Bouwmeester et al. (T. Bouwmeester et al., 1996,
Nature 382: 595-601), the sequence of mouse DAN is as described by Ozaki et al. (T.
Ozaki et al., 1996, Jpn. J. Cancer Res. 87: 58-61). (B) A table summarizing the degree of identity of amino acid sequences of homologous regions of “PRDC”, mouse DAN, and Xenopus Cerberus.

FIG. 6 shows the results of Northern blot analysis of “PRDC” mRNA. E11.5 embryo wild type (lane 1), hetero (lane 2), and homo (lane 3) poly (A) + RNA
(About 2.5 μg) was electrophoresed on a 1% denaturing agarose gel, transferred to a nylon membrane, and then hybridized with a PRDC cDNA probe. A signal of about 3.7 kb was detected in each sample. A GAPDH (glyceraldehyde-3-phosphate dehydrogenase) probe was used as a control to compare the amount of RNA migrated.

FIG. 7 shows embryos generated by injecting (A) “PRDC” mRNA or (B) lacZ mRNA into Xenopus embryos by microinjection.

FIG. 8 (A) and (B) show “PRDC” mRNA and lacZ mRN.
FIG. 2 is a view showing embryos which were both injected into Xenopus laevis embryos by microinjection and developed, and then subjected to X-gal staining.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 16/18 C12N 1/19 C12N 1/19 1/21 1/21 C12P 21/02 C 5/10 C12Q 1/68 C12P 21/02 A61K 37/02 C12Q 1/68 C12N 5/00 B // (C12N 5/10 C12R 1:91)

Claims (10)

[Claims] 1. A protein comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein, and induces differentiation of brain nerve tissue A protein having the activity of 2. It consists of the nucleotide sequence of SEQ ID NO: 2.
A protein encoded by a DNA that hybridizes with DNA, the protein having an activity of inducing differentiation of brain nerve tissue. 3. A DNA encoding the protein according to claim 1 or 2. A vector into which the DNA according to claim 3 has been inserted. 5. A transformant which retains the DNA according to claim 3 so that it can be expressed. 6. The method for producing the protein according to claim 1, comprising a step of culturing the transformant according to claim 5. 7. An antibody that binds to the protein according to claim 1 or 2. 8. It consists of the nucleotide sequence of SEQ ID NO: 2.
DNA that specifically hybridizes to DNA and has a chain length of at least 15 nucleotides. 9. A transgenic mouse that expressively retains a DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1. 10. A knockout mouse in which a genomic DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 has been modified or destroyed.