Patent Application
20100028866
The present invention relates to a Nato3 gene, which is a dopaminergic neuron proliferative progenitor cell marker. More particularly, the present invention relates to a means for detecting a dopaminergic neuron proliferative progenitor cell, a method for detecting the cell, and a kit for detecting the cell.
The dopamine system is a very important system involved in movement control, hormone secretion control, affectivity control, and so forth, which are important in the mammalian brain. Therefore, abnormalities in dopaminergic neurotransmission cause various disorders of the neural system. For example, the Parkinson's disease is a neurodegenerative disease of the extrapyramidal system which is caused by specific degeneration of dopaminergic neurons in the midbrain substantia nigra (HARRISON'S PRINCIPLES OF INTERNAL MEDICINE Vol. 2 23rd ed., Isselbacher et al. edited by McGraw-Hill Inc., NY (1994) pp. 2275-7).
As a method for treating the Parkinson's disease, a method of orally administering L-DOPA (3,4-dihydroxy-phenylalanine) has been mainly adopted for compensating the decrease in the amount of the produced dopamine, but it is known that the duration of the effect is not good.
Accordingly, as a method for compensating the loss of dopaminergic neurons, recently, there has been attempted a therapeutic method of transplanting a midbrain ventral region of a 6-9 week aborted fetus containing dopaminergic neuron precursors (U.S. Pat. No. 5,690,927; Spencer et al. (1992) N. Engl. J. Med. 327:1541-8; Freed et al. (1992) N. Engl. J. Med. 327:1549-55; Widner et al. (1992) N. Engl. J. Med. 327:1556-63; Kordower et al. (1995) N. Engl. J. Med. 332:1118-24; Defer et al. (1996) Brain 119:41-50; and Lopez-Lozano et al. (1997) Transp. Proc. 29:977-80). However, at the present time, in addition to cell supply and ethical issues (Rosenstain (1995) Exp. Neurol. 33:106; Turner et al. (1993) Neurosurg. 33:1031-7), various other problems have been indicated, for example, risk of infectious contamination, immunologic transplant rejection (Lopez-Lozano et al. (1997) Transp. Proc. 29:977-80 and Widner and Brudin (1988) Brain Res. Rev. 13:287-324), low survival rate due to the fetus tissue's mainly dependence on lipid metabolism rather than glycolysis (Rosenstein (1995) Exp. Neurol. 33:106), and so forth.
As a method for solving the problem of the ethical issues or supply shortage, for example, a method by using a cortex, a striation, and midbrain cells, derived from a pig, and so forth have been proposed (for example, Japanese Patent Laid-Open Publication No. 10-508487, No. 10-508488, and No. 10-509034). However, in this method, a complex procedure for modifying an antigen on the cell surface (MHC class I antigen) is required to suppress rejection. As a method for solving the transplant rejection, for example, a method involving local immunosuppression by simultaneously transplanting Sertoli cells has been proposed (Japanese Patent Laid-Open Publication No. 11-509170 and No. 11-501818; and Selawly and Cameron (1993) Cell Transplant 2:123-9). It is possible that transplant cells are obtained from a relative whose MHC matches, bone marrow of another person, a bone marrow bank, a cord blood bank, and so forth. However, if patient's own cells can be used, the problems of rejection can be solved without extra procedures and trouble.
Accordingly, it has been expected that, instead of cells derived from an aborted fetus, a differentiation system of dopaminergic neurons in vitro from non-neural cells such as embryo-stem (ES) cell and bone marrow stromal cells are utilized as a transplant material. Actually, there is a report that a functional dopaminergic neuron is formed by ES cell transplantation into lesion striation of a rat Parkinson's disease model (Kim et al. (2002) Nature 418:50-56). It is thought that in the future, importance of regenerative medicine from ES cells or the patient's own neural stem cells will increase.
On the other hand, in the treatment of damage of neural tissue, restructuring of brain function is required, and for forming appropriate linkage with surrounding cells (network formation), not mature cells but progenitor cells that can differentiate into neurons in vivo are required to be transplanted. However, in the transplantation of neuron progenitor cells, in addition to the above-described problem regarding supply, there is a problem that the progenitor cells can differentiate into a nonuniform cell population. For example, in the treatment of the Parkinson's disease, it is necessary that dopaminergic neurons are selectively transplanted among catecholamine-containing neurons. Before now, as transplant cells for use in the treatment of the Parkinson's disease, there has been proposed a striate body (Lindvall et al. (1989) Arch. Neurol. 46:615-31 and Widner et al. (1992) N. Engl. J. Med. 327:1556-63), an immortalized cell line derived from human embryonic nerve (Japanese Patent Laid-Open Publication No. 8-509215, No. 11-506930, and No. 2002-522070), a post-mitotic human neuron of NT2Z cells (Japanese Patent Laid-Open Publication No. 9-5050554), a neuron primordial cell (Japanese Patent Laid-Open Publication No. 11-509729), a cell transfected with an exogenous gene so as to produce catecholamine such as dopamine, a bone marrow stromal cell (Japanese Patent Laid-Open Publication No. 2002-504503 and No. 2002-513545), an ES cell in which a gene is modified (Kim et al. (2002) Nature 418:50-56), and so forth. However, none of these contain only dopaminergic neurons or cells to differentiate into dopaminergic neurons.
As a method for selectively condensing or isolating dopaminergic neurons from undifferentiated cell population, there has been proposed a method of, introducing a reporter gene expressing a fluorescent protein under control of promoter/enhancer of a gene such as tyrosine hydroxylase (TH) expressed in dopaminergic neurons into each cell of the cell population, isolating the cells emitting fluorescence, and thereby visualizing the alive dopaminergic neurons to condense, segregate or identify (Japanese Patent Laid-Open Publication No. 2002-51775). However, this method requires a complex step of introduction of an exogenous gene, and furthermore, when used in gene treatment, the existence of the reporter gene causes problem of toxicity and immunogenicity.
As described above, now, one of the largest problems in transplantation treatment for the Parkinson's disease is that the either dopaminergic neuron progenitor cells derived from the midbrain ventral region of aborted fetus or induced to differentiate are a mixture of various cells. It is desirable that only a desired cell species is isolated and used, considering safety in neural network formation. Furthermore, considering survival or ability for correctly forming a network in a brain in which the cells are transplanted, it can be said that it is desirable from the treatment effect that earlier proliferative progenitor cells are isolated and transplanted.
Before now, as a gene that selectively expresses in the dopaminergic neuron proliferative progenitor cells, Lrp4 (WO 2004/065599) has been reported. Additionally, some markers of dopaminergic neuron progenitor cells have been reported (WO 2004/038018 and WO 2004/052190). Among them, with respect to Lmx1a, expression has been confirmed in human and mouse dopaminergic neuron proliferative progenitor cells, postmitotic dopaminergic neuron precursor cells, and dopaminergic neurons (WO 2005/052190).
By the way, the Nato3 gene is a transcription factor having a basic helix-loop-helix (bHLH), and it has been reported that the Nato3 gene is expressed in the midbrain, and has an important role in the developmental process (Segev, E., N. Halachmi, A. Salzberg, and N. Ben-Arie. 2001. Mech Dev 106:197-202). Moreover, it has been reported that Nato3 is expressed in the midbrain, particularly, the most ventral side (floor plate), and relates to differentiation of neural cells (Verzi, M. P., J. P. Anderson, E. Dodou, K. K. Kelly, S. B. Greene, B. J. North, R. M. Cripps, and B. L. Black. 2002. Dev Biol 249:174-90).
However, it is not reported that Nato3 is selectively expressed in dopaminergic neuron proliferative progenitor cells.
The present inventors have recently found that a Nato3 gene (hereinafter, occasionally referred to as merely “Nato3”) is selectively expressed in dopaminergic neuron proliferative progenitor cells. The present invention is based on this finding.
An object of the present invention is to provide a means for detecting a dopaminergic neuron proliferative progenitor cell, a method for detecting a dopaminergic neuron proliferative progenitor cell, and a kit for detecting a dopaminergic neuron proliferative progenitor cell.
Further, an object of the present invention is to provide a method for screening for a substance effective for inducing differentiation into dopaminergic neuron proliferative progenitor cells.
Furthermore, an object of the present invention is to provide a method for producing a dopaminergic neuron proliferative progenitor cell for use in the treatment of the Parkinson's disease.
The present invention provides a polynucleotide probe or polynucleotide primer for use in the detection or selection of a dopaminergic neuron proliferative progenitor cell, which can hybridize with a polynucleotide consisting of a nucleotide sequence of a Nato3 gene, or a complementary sequence thereto (hereinafter, occasionally referred to as “probe according to the present invention” or “primer according to the present invention”).
The present invention provides an antibody against a Nato3 protein, or a part thereof for use in the detection or selection of a dopaminergic neuron proliferative progenitor cell (hereinafter, occasionally referred to as “antibody according to the present invention”).
The present invention also provides a method for detecting or selecting a dopaminergic neuron proliferative progenitor cell, comprising the step of detecting expression of a Nato3 gene (hereinafter, occasionally referred to as “detection method according to the present invention”).
The present invention further provides a kit for detecting or selecting a dopaminergic neuron proliferative progenitor cell comprising at least a probe according to the present invention, a primer according to the present invention, or an antibody according to the present invention.
The present invention provides a method for screening for a substance effective for inducing differentiation into a dopaminergic neuron proliferative progenitor cell, comprising the step of detecting expression of a Nato3 gene.
The present invention provides a method for producing a dopaminergic neuron proliferative progenitor cell for use in the treatment of the Parkinson's disease.
The present invention provides use of a polynucleotide that can hybridize with a polynucleotide consisting of a nucleotide sequence of a Nato3 gene, or a complementary sequence thereto, for detecting or selecting a dopaminergic neuron proliferative progenitor cell.
The present invention provides use of an antibody against a Nato3 protein, or a part thereof, for detecting or selecting a dopaminergic neuron proliferative progenitor cell.
The probe according to the present invention, the primer according to the present invention, and the antibody according to the present invention can be utilized as selective markers for dopaminergic neuron proliferative progenitor cells. Accordingly, the present invention is extremely useful in a purity test of a transplant material and development of a method for inducing differentiation into dopaminergic neuron proliferative progenitor cells in vitro, or the like, and is expected to contribute to the promotion of practical application of regenerative medicine.
Hereinafter, the present invention will be explained in detail. The following description is an example for explaining the present invention and the present invention is not limited to the embodiments to be described. All technical terms, scientific terms, and terminologies used in the present specification have the same meanings as those that are generally understood by those ordinary skilled in the art in the technical fields to which the present invention belongs and are used merely for the purpose of explanation of a specific embodiment but are not intended to make limitation. The present invention can be carried out in various embodiments as long as not departing from the spirit thereof. All the prior art documents, published publications, patent publications, and other patent documents, cited in the present specification, are incorporated into the present specification as references, and can be used for carrying out the present invention.
The “dopaminergic neuron proliferative progenitor cell”, which is an object to be detected or selected in the present invention, means a dopaminergic neuron progenitor cell before arrest of mitotic division.
The dopaminergic neuron differentiates from a neuroepithelial cell, through the differentiation stages of a proliferative progenitor cell and a postmitotic precursor cell, into a mature dopaminergic neuron. The dopaminergic neuron proliferative progenitor cell is the earliest progenitor cell in the dopaminergic neurons, and therefore, high survival rate and high ability of network formation in the brain to which the cell is transplanted can be expected. Therefore, the dopaminergic neuron proliferative progenitor cell is useful for transplantation therapy for diseases caused by decrease in dopamine due to degeneration of the dopaminergic neurons such as the Parkinson's disease.
The cells selected by using the probe, the primer, or the antibody according to the present invention as an index are dopaminergic neuron proliferative progenitor cells before arrest of mitotic division, and therefore, are preferable for the transplantation treatment for neurodegenerative diseases such as the Parkinson's disease in the aspects of safety, survival rate, and network formation ability, compared to a conventional mixed cell population or dopaminergic neuron progenitor cells in which an exogenous gene is introduced. The cells are dopaminergic neuron proliferative progenitor cells before arrest of mitotic division, namely, in proliferation, and have the possibility of differentiating to mature in the most appropriate place in the brain, and also, the dopaminergic neuron progenitor cells have the possibility of proliferating in vivo, and therefore, a longer effect of the treatment can be expected. Therefore, it can be said that the present invention paves the way to the practical application of the effective transplantation treatment of neurodegenerative diseases such as the Parkinson's disease.
The probe and the primer according to the present invention can hybridize specifically with a Nato3 gene. As described above, the expression of a Nato3 gene is useful as an index of dopaminergic neuron proliferative progenitor cells. Therefore, the probe or the primer according to the present invention can be used as a marker for detecting dopaminergic neuron proliferative progenitor cells.
The probe and the primer according to the present invention can be used for detecting expression of a Nato3 gene, and corresponds to a polymer consisting of a plurality of bases or base pairs such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It is known that double-strand cDNA can also be used in tissue in situ hybridization, and such double strand cDNA is included in the probe and the primer according to the present invention. As a particularly preferable probe and primer in detection of RNA in tissue, an RNA probe (riboprobe) can be exemplified.
The probe and the primer according to the present invention includes those consisting of a polynucleotide comprising a sequence of at least 10, preferably at least 15, more preferably at least 20, and further preferably at least 25 contiguous nucleotides of a nucleotide sequence of a Nato3 gene, or a complementary sequence thereto. Also, the probe and the primer according to the present invention includes those consisting of a polynucleotide comprising a sequence of 10-50 or 10-30, 15-50 or 15-30, 20-50 or 20-30, and 25-50 or 25-30 contiguous nucleotides of a nucleotide sequence of a Nato3 gene, or a complementary sequence thereto.
The probe and the primer according to the present invention can be at least 10 base length, preferably at least 15 base length, more preferably at least 20 base length, further preferably at least 25 base length. The probe and the primer according to the present invention can also be 10-50 base length or 10-30 base length, 15-50 base length or 15-30 base length, 20-50 base length or 20-30 base length, and 25-50 base length or 25-30 base length.
Preferable embodiments of the probe and the primer according to the present invention provide a probe and a primer having 15-30 base length for use in the detection or selection of a dopaminergic neuron proliferative progenitor cell, consisting of a polynucleotide comprising a sequence of at least 10 (preferably at least 15, more preferably at least 20, and further preferably at least 25) contiguous nucleotides of a nucleotide sequence of a Nato3 gene, or a complementary sequence thereto, which can hybridize with a Nato3 gene.
Preferable embodiments of the probe and the primer according to the present invention provide those that can hybridize with a high discrimination part in the nucleotide sequence of a Nato3 gene. By using such a probe and a primer, it becomes possible to detect the proliferative progenitor cells with higher accuracy. Such a probe and a primer include those that can hybridize with a nucleotide sequence comprising a part or all of a nucleotide sequence selected from the group consisting of nucleotides 1-365 and 534-640 of SEQ ID NO:1, nucleotides 1-376 and 545-662 of SEQ ID NO:3, nucleotides 1-306 and 475-501 of SEQ ID NO:5, nucleotides 1-377 and 546-886 of SEQ ID NO:7, nucleotides 1-312 and 481-507 of SEQ ID NO:9, nucleotides 1-306 and 475-501 of SEQ ID NO:11, nucleotides 1-306 and 475-501 of SEQ ID NO:13, nucleotides 1-177 and 346-372 of SEQ ID NO:15, nucleotides 1-359 and 528-557 of SEQ ID NO:17, nucleotides 1-357 and 523-552 of SEQ ID NO:19.
The probe according to the present invention can be used as a probe according to the general methods in known methods for detecting a gene of interest such as a northern blotting method, a southern blotting method, an in situ hybridization method, and so forth.
The probe according to the present invention can be chemically synthesized based on the nucleotide sequences disclosed in the present specification. The preparation of the probe is well-known and can be performed, for example, according to “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989)) and “Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997)).
The primer according to the present invention can also be used as a primer set consisting of two or more kinds of the primers.
The primer and the primer set according to the present invention can be utilized as a primer and a primer set according to the general methods in known methods for detecting a gene of interest by utilizing a nucleic acid amplification method such as a PCR method, a RT-PCR method, a real-time PCR method, an in situ PCR method, or a LAMP method.
The primer set according to the present invention can be selected so that the nucleotide sequence of a Nato3 gene can be amplified by a nucleic acid amplification method. Nucleic acid amplification methods are well-known, and selection of the primer pair in the nucleic acid amplification method is understood by those skilled in the art. For example, in the PCR method, primers can be selected so that one of the two primers (primer pair) is paired with the plus strand of the double strand DNA of a Nato3 gene, the other primer is paired with the minus strand of the double strand DNA, and with a strand extended by one primer, the other primer can be paired. Moreover, in the LAMP method (WO 00/28082), with respect to the target gene, three regions F3c, F2c, and F1c are defined from the 3′ end side, and three regions B1, B2, and B3 are defined from the 5′ end side, and by using the six regions, four kinds of primers can be designed.
The primer according to the present invention can be chemically synthesized based on the nucleotide sequences disclosed in the present specification. The preparation of the probe is well-known and can be performed, for example, according to “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989)), “Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997)).
In the present invention, the “Nato3 gene”, which is an index of the existence of the dopaminergic neuron proliferative progenitor cell, is known in human, mouse, rat, chimpanzee, dog, bovine, chicken, and so forth. GenBank Accession Numbers disclosing the respective sequences are as follows.
Human: NM—152898 (SEQ ID NO:1 (base sequence), SEQ ID NO:2 (amino-acid sequence), hereinafter representation will be in the same order), AF517122 (SEQ ID NO:3, SEQ ID NO:4), BC069147(SEQ ID NO:5, SEQ ID NO:6), AF369897 (same as BC069147)
Mouse: NM—033522(SEQ ID NO:7, SEQ ID NO:8), AF517121(same as NM—033522), AF369896(SEQ ID NO:9, SEQ ID NO:10)
Also with respect to an animal (preferably mammal) except for the above-described animals, those skilled in the art can specify a sequence of a Nato3 gene inherent in the animal, based on the known full-length sequence of a Nato3 gene. For example, by homology search based on the human or mouse Nato3 gene, a Nato3 gene of the animal can be searched and identified. In the homology search, BLAST to be described later or the like can be used.
The Nato3 gene includes:
a polynucleotide encoding a human Nato3 protein consisting of an amino acid sequence of at least one selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6;
a polynucleotide encoding a mouse Nato3 protein consisting of an amino acid sequence of at least one selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:10;
a polynucleotide encoding a rat Nato3 protein consisting of an amino acid sequence of SEQ ID NO:12;
a polynucleotide encoding a chimpanzee Nato3 protein consisting of an amino acid sequence of SEQ ID NO:14;
a polynucleotide encoding a dog Nato3 protein consisting of an amino acid sequence of SEQ ID NO:16;
a polynucleotide encoding a bovine Nato3 protein consisting of an amino acid sequence of SEQ ID NO: 18; and
a polynucleotide encoding a chicken Nato3 protein consisting of an amino acid sequence of SEQ ID NO:20.
Moreover, the Nato3 gene includes:
a polynucleotide comprising a human Nato3 gene DNA sequence of at least one selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5;
a polynucleotide comprising a mouse Nato3 gene DNA sequence of at least one selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:9;
a polynucleotide comprising a rat Nato3 gene DNA sequence of SEQ ID NO:11;
a polynucleotide comprising a chimpanzee Nato3 gene DNA sequence of SEQ ID NO:13;
a polynucleotide comprising a dog Nato3 gene DNA sequence of SEQ ID NO:15;
a polynucleotide comprising a bovine Nato3 gene DNA sequence of SEQ ID NO: 17; and
a polynucleotide comprising a chicken Nato3 gene DNA sequence of SEQ ID NO:19.
The Nato3 gene according to the present invention includes genes encoding proteins which are functionally equivalent to a Nato3 protein. Whether the gene is “functionally equivalent” can be determined by evaluating a biological phenomenon or function relating to the expression of a Nato3 gene, for example, by evaluating whether the gene is selectively expressed in a dopaminergic neuron proliferative progenitor cell.
Moreover, the proteins which are functionally equivalent to a Nato3 protein include proteins having polymorphism when the gene encoding the amino acid sequence (such as amino acid sequence of SEQ ID NO:2) has polymorphism.
The gene encoding the proteins which are functionally equivalent to the Nato3 protein includes:
a gene encoding an amino acid sequence (modified amino acid sequence) of a Nato3 protein (for example, the amino acid sequence of SEQ ID NO:2 and the amino acid sequence of SEQ ID NO:8) in which one or more amino acid residues are inserted, substituted or deleted, or are added to one or both ends in the amino acid sequence;
a gene that can hybridize under stringent conditions with a gene encoding an amino acid sequence of a Nato3 protein (for example, the amino acid sequence of SEQ ID NO:2 and the amino acid sequence of SEQ ID NO:8); and
a gene encoding an amino acid sequence having at least an identity of 70% or more with an amino acid sequence of a Nato3 protein (for example, the amino acid sequence of SEQ ID NO:2 and the amino acid sequence of SEQ ID NO:8).
In the present specification, “one or more amino acid residues are inserted, substituted or deleted, or are added to one or both ends in the amino acid sequence” means that the modification is performed by a well-known technical method such as a site-directed mutagenesis or by substitution of a plurality of some amino acids to an extent of being naturally generated, or the like.
The modified amino acid sequence of a Nato3 protein can be an amino acid sequence in which, for example, 1-30, preferably 1-20, more preferably 1-9, further preferably 1-5, and particularly preferably 1 or 2 amino acid(s) is/are inserted, substituted, or deleted, or is/are added to one or both of end(s) in amino acid sequence. The modified amino acid sequence can be preferably an amino acid sequence having one or more (preferably, one or several, or 1, 2, 3 or 4) conservative substitutions in the amino acid sequence of the Nato3 protein.
The term “conservative substitutions” means that one or more amino acid residues are substituted with other chemically analogous amino acid residues so as not to substantially change protein activity. For example, the case that a certain hydrophobic residue is substituted with another hydrophobic residue and the case that a certain polar residue is substituted with another polar residue having the same charge can be exemplified. Functionally analogous amino acids which can be substituted in such a manner are known in the technical field, with respect to every amino acid. To give specific examples, the non-polar (hydrophobic) amino acid includes alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine. The polar (neutral) amino acid includes glycine, serine, threonine, tyrosine, glutamine, asparagines, cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Moreover, negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
In the present specification, “hybridize” means hybridization to a target polynucleotide under stringent conditions. Specifically, there can be exemplified a polynucleotide having identity of at least 70% or more, preferably 80% or more, more preferably 85% or more, further preferably 90% or more, further more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more, with the target nucleotide sequence when calculation is performed using a parameter of default (initial setting) with homology search software such as FASTA, BLAST, Smith-Waterman [Meth. Enzym., 164, 765 (1988)]. Moreover, “under stringent conditions” can be performed according to a method of performing reaction in a hybridization buffer solution that can be generally used by those skilled in the art so that the temperature is 40-70° C., and preferably 60-65° C. and performing rinsing in a rinse solution whose salt concentration is 15-300 mmol/L, and preferably 15-60 mmol/L. The temperature and the salt concentration can be appropriately adjusted according to length of the probe to be used. Furthermore, the condition when the hybridized nucleotide is rinsed can be 0.2 or 2×SSC, 0.1% SDS, and a temperature of 20-68° C. As to control of the stringent conditions (high stringency) or the mild condition (low stringency), the difference can be provided by salt concentration or temperature in rinsing. When the difference of the hybridization is provided by salt concentration, a stringent wash buffer (high stringency wash buffer) of 0.2×SSC and 0.1% SDS can be used, and a mild wash buffer (low stringency wash buffer) of 2×SSC and 0.1% SDS. Moreover, when the difference of the hybridization is provided by temperature, the temperature is 68° C. in the stringent case, 42° C. in the case of moderate stringency, and room temperature (20-25° C.) in the mild case, and every case thereof may be performed under 0.2×SSC and 0.1% SDS.
In general, the prehybridization is performed under the same conditions as the hybridization. However, hybridization and preliminary rinsing are not limited to be performed under the same conditions.
The hybridization can be performed according to a known method. Moreover, in the case of using a commercially available library, the hybridization can be performed according to the method described in the appended instruction for use.
In the present specification, the term “identity” (occasionally referred to as homology) with respect to amino acid sequences means the degree of identity of the amino acid residues of the respective sequences between the sequences to be compared. In this case, existence of a gap and property of the amino acid are considered (Wilbur, Natl. Acad. Sci. U.S.A. 80:726-730 (1993)). For calculation of the homology, BLAST (Altschul: J. Mol. Biol. 215:403-410 (1990)), FASTA (Peasron: Methods in Enzymiology 183:63-69 (1990)), or the like can be used.
The amino acid sequence having at least an identity of 70% or more with the amino acid sequence of a Nato3 protein can be an amino acid sequence having identity of preferably 80% or more, more preferably 85% or more, further preferably 90% or more, further more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.
The “identity” may be a value calculated by using a homology search program known by those skilled in the art and can be calculated, for example, by using a parameter of default (initial setting) in the homology algorithm BLAST(Basic local alignment search tool) http:www.ncbi.nlm.nih.gov/BLAST/ in NCBI (National Center for Biotechnology Information).
The antibody according to the present invention can recognize specifically a Nato3 protein. As described above, the expression of a Nato3 gene is useful as an index for dopaminergic neuron proliferative progenitor cells. Therefore, the antibody according to the present invention can be used as a marker for detecting dopaminergic neuron proliferative progenitor cells.
A Nato3 protein for obtaining the antibody according to the present invention may have antigenicity of Nato3 and includes a protein in which one or more amino acid residues are deleted, inserted, substituted, or added in an amino acid sequence of a Nato3 protein. It is known that in such a protein, the same biological activity as the original protein is maintained (Mark et al. (1984) Proc. Natl. Acad. Sci. USA 81:5662-6; Zoller and Smith (1982) Nucleic Acids Res. 10:6487-500; Wang et al. (1984) Science 224:1431-3; and Dalbadie-McFarland et al. (1982) Proc. Natl. Acad. Sci. USA 79:6409-13). A method that one or more amino acid residues are deleted, inserted, substituted, or added in the state of maintaining the antigenicity of the original protein in a protein is known. For example, a polynucleotide encoding a mutant protein can be prepared by a site-directed mutagenesis and can be appropriately expressed (Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997); Sections 8.1-8.5; Hashimoto-Goto et al. (1995) Gene 152:271-5; Kinkel (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kramer and Fritz (1987) Method. Enzymol. 154:350-67; and Kunkel (1988) Method. Enzymol. 85:2763-6).
The antibody according to the present invention includes an antibody specific to a part of a Nato3 protein. Specifically, a Nato3 protein for obtaining an antibody of the present invention includes a polypeptide fragment having at least 6 amino acid residues or more (for example, 6, 8, 10, 12, or 15 amino acid residues or more) of the Nato3 protein, as well as a polypeptide having a full-length amino acid sequence. The polypeptide fragment of the Nato3 protein in the present specification includes every fragment as long as the fragment has antigenicity of the Nato3 protein.
The preferable fragment includes polypeptide fragments such as the amino terminal or the carboxyl terminal of the Nato3 protein. The antigenic determinant site of the polypeptide is estimated by a method of analyzing hydrophobicity/hydrophilicity of the amino acid sequence of the protein (Kyte-Doolittle(1982) J. Mol. Biol. 157:105-22) or a method of analyzing the secondary structure (Chou-Fasman (1978) Ann. Rev. Biochem. 47:251-76), and furthermore, confirmed by a computer program (Anal. Biochem. 151:540-6 (1985)) or a technique such as a PEPSCAN method (Japanese Patent Laid-Open Publication No. 60-500684) of synthesizing a short peptide and confirming the antigenicity.
The antibody against the Nato3 protein includes:
an antibody against a protein having an amino acid sequence of at least one selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, or a part thereof;
an antibody against a protein having an amino acid sequence of at least one selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:10, or a part thereof;
an antibody against a protein having an amino acid sequence of SEQ ID NO: 12, or a part thereof;
an antibody against a protein having an amino acid sequence of SEQ ID NO: 14, or a part thereof;
an antibody against a protein having an amino acid sequence of SEQ ID NO: 16, or a part thereof;
an antibody against a protein having an amino acid sequence of SEQ ID NO: 18, or a part thereof; and
an antibody against a protein having an amino acid sequence of SEQ ID NO: 20, or a part thereof.
Preferable embodiments of the antibody according to the present invention provide an antibody recognizing a high discrimination polypeptide portion in a Nato3 protein. By using such an antibody, it becomes possible that the proliferative progenitor cells can be detected with higher accuracy. Such antibody includes an antibody against a high discrimination polypeptide portion in a Nato3 protein, for example, at least 6 amino acid residues or all of an amino acid sequence selected from a group consisting of amino acids 1-102 and 159-166 of SEQ ID NO:2, amino acids 1-102 and 159-166 of SEQ ID NO:4, amino acids 1-102 and 159-166 of SEQ ID NO:6, amino acids 1-104 and 161-168 of SEQ ID NO:8, amino acids 1-104 and 161-168 of SEQ ID NO:10, amino acids 1-102 and 159-166 of SEQ ID NO:12, amino acids 1-102 and 159-166 of SEQ ID NO:14, amino acids 1-59 and 116-123 of SEQ ID NO:16, amino acids 1-101 and 158-164 of SEQ ID NO:18, amino acids 1-119 and 176-183 of SEQ ID NO:20.
The antibody according to the present invention can also be obtained by using a well-known method for those skilled in the art (for example, “Current Protocols in Molecular Biology” (John Wiley & Sons (1987) and Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988).
The antibody according to the present invention includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single-strand antibody (scFv), a humanized antibody, a polyspecific antibody, and antibody fragments such as Fab, Fab′, F(ab′)2, Fc, and Fv.
In the case of the polyclonal antibody, the blood of a mammal in which an antigen is sensitized is extracted and serum is segregated from the blood by a known method to serve as the serum containing the polyclonal antibody.
According to need, fractions containing the polyclonal antibodies can also be further isolated.
In the case of the monoclonal antibody, antibody-producing cells obtained from the spleen or the lymph node of a mammal in which the above-described antigen is sensitized are extracted and cell-fused with myeloma cells. The obtained hybridoma is cloned and the antibody is collected from the culture to serve as the monoclonal antibody.
As the immunizing antigen, a fragment of the Nato3 protein can be used. Alternatively, an antigen synthesized based on the above-described amino acid sequence can be used. The antigen may be used as a complex with a carrier protein. For preparation of the complex of the antigen and the carrier protein, various condensation agents such as glutaraldehyde, carbodiimide, maleimide-activated ester, or the like can be used. The carrier protein may be one generally used such as bovine serum albumin, thyroglobulin, hemocyanin, or the like and is generally performed coupling at a ratio of 1-5.
The animal to be immunized includes mouse, rat, rabbit, guinea pig, and hamster. The injection method includes subcutaneous, muscular, or intraperitoneal administration. In the administration, the antigen may be mixed with complete Freund's adjuvant or incomplete Freund's adjuvant. The administration is generally performed one time per 2-5 weeks.
The antigen-producing cells obtained from the spleen or the lymph node of the immunized animal is cell-fused with myeloma cells and isolated as hybridomas. The myeloma cells derived from mouse, rat, or human are used, and are preferably derived from the same species as the antigen-producing cells, but cells between different species are occasionally possible.
The cell fusion can be performed according to a previously known method, for example, the method disclosed in Nature, 256, 495, 1975.
The fusion accelerator includes polyethylene glycol or Sendai virus, and in general, the cell fusion can be performed by reaction for approximately 1-10 minutes so that the ratio of the number of the antigen-producing cells and the number of the myeloma cells is generally approximately 1:1-10:1, under a temperature of 20-40° C., and preferably 30-37° C. by using polyethylene glycol (average molecular weight 1000-4000) having a concentration of approximately 20-50%.
For the screening of the antigen-producing hybridoma, various immunochemical methods can be used. For example, an ELISA method in which a microplate on which the Nato3 protein is coated is used, an EIA method in which a microplate on which an anti-immunoglobulin antibody is coated is used, and an immunoblotting method in which a nitrocellulose transfer membrane is used after electrophoresing samples containing the Nato3 protein.
From such wells, further cloning is performed by, for example, a limiting dilution method, and thereby, clones can be obtained. Selection and breeding of hybridomas are generally performed in a medium for animal cells (for example, RPMI1640) containing 10-20% bovine embryo serum to which HAT (hypoxanthine, aminopterin, and thymidine) is added. The clones obtained as described above are transplanted into the abdominal cavity of a SCID mouse to which pristine is preliminarily administered, and ascitic fluid containing the monoclonal antibody at high concentration is collected after 10-14 days to serve as a material for antibody purification. Also, the clones can be cultured and the culture can be a material for antibody purification.
For the purification of the monoclonal antibody, a previously known method may be used as the purification method of immunoglobulin, and the purification can be easily achieved, for example, by an ammonium sulfate fraction method, a PEG fraction method, an ethanol fraction method, utilization of an anion exchanger, affinity chromatography in which a Nato3 protein is used, or the like.
The purification of the polyclonal antibody from the serum can be performed similarly.
The expression of a Nato3 gene serves as an index of the existence of dopaminergic neuron proliferative progenitor cells. Therefore, according to the present invention, by detecting expression of a Nato3 gene, the dopaminergic neuron proliferative progenitor cells can be detected or selected.
The method for “detecting expression of a Nato3 gene” used herein is not particularly limited as long as being capable of detecting the expression of a Nato3 gene in the cell samples to be tested, and, for example, includes hybridization methods, nucleic acid amplification methods, and antigen-antibody reaction methods.
The “cell samples to be tested” used herein can be cell samples that are thought to contain the dopaminergic neuron proliferative progenitor cells, and the cells in the midbrain ventral region can be used. The cells in the midbrain ventral region can be obtained by a known method (Studer, L., et al. Nature Neurosci (1998) 1:290-295). Preferably, fetus' (preferably, human aborted fetus') or the patient's own cells of the midbrain ventral region can be used as the cell samples to be tested. Moreover, the culture cells containing dopaminergic neuron proliferative progenitor cells induced to differentiate in vitro can be used as the cell samples to be tested. The induction to differentiate into the dopaminergic neuron proliferative progenitor cells in vitro can be performed by the differentiation treatment by a known method such as an SDIA method (Kawasaki et al. Neuron (2000) 28(1):31-40) or a 5-stage method (Lee, S H., et al. Nature Biotech (2000) 18:675-579) by using, as a starting material, known ES cells (Kawasaki et al. Neuron (2000) 28(1):31-40 and Lee, S H., et al. Nature Biotech (2000) 18:675-579), bone marrow stromal cells, immortalized cell line derived from nerve (Japanese Patent Laid-Open Publication No. 8-509215, No. 11-506930, and No. 2002-522070), neuron primordial cells (Japanese Patent Laid-Open Publication No. 11-509729), or the like. Preferably, ES cells subjected to the differentiation treatment by the SDIA method can be used as the cell samples to be tested.
The “SDIA method” used herein can be performed by co-culturing the ES cells and the stromal cell line PA6 in a serum-free medium (Kawasaki et al. Neuron (2000) 28(1):31-40). Moreover, the “5-stage method” can be performed as follows. ES cells are cultured on a non-adherent culture plate under existence of the serum and thereby an embryoid body (EB) is formed, and sequentially, the EB is attached onto an adherent culture plate, and thereby, the neuron progenitor cells are selected. Finally, a growth factor such as Shh, FGF2, or FGF8 is added thereto, and thereby, dopaminergic neuron progenitor cells are induced (Lee, SH., et al. Nature Biotech (2000) 18:675-579).
According to the first embodiment of the detection method according to the present invention, the probe according to the present invention hybridizes with a nucleic acid sample (mRNA or transcript thereof), and the hybridization complex, namely, nucleotide double strand, is detected. Thus, the expression of the Nato3 gene can be detected in the cell samples.
For the detailed procedure of the hybridization method, there can be referred to “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989), particularly Sections 9.47-9.58, “Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997)), particularly, Sections 6.3 and 6.4, “DNA Cloning 1: Core Techniques, A Practical Approach 2nd ed.” (Oxford University (1995), particularly, Section 2.10 for the conditions).
The detection of expression of the Nato3 gene by utilizing the hybridization method can be performed, for example, by the following steps of:
(a) contacting a polynucleotide derived from a cell sample to be tested, with the polynucleotide probe according to the present invention; and
(b) detecting a hybridization complex.
In step (a), mRNA prepared from the cell sample to be tested that is thought to contain dopaminergic neuron proliferative progenitor cells or complementary DNA (cDNA) transcribed from the mRNA, as the polynucleotide derived from the cell sample to be tested, can be contacted with the probe.
In the detection method by using a probe, the probe can be labeled. The label includes a label by utilizing radioactivity (such as 32P, 14C, and 35S), fluorescence (such as FITC, europium), an enzyme (such as peroxidase or alkaline phosphatase) reaction such as chemical coloring, or the like.
The detection of the hybridization product can be performed by using a well-known method such as northern hybridization, southern hybridization, or colony hybridization.
The cells in which the hybridization complex is detected are those expressing a Nato3 gene, and therefore can be determined as the dopaminergic neuron proliferative progenitor cells.
According to the second embodiment of the detection method according to the present invention, the expression of the Nato3 gene can be detected in the cell sample by amplifying nucleic acid samples (mRNA or transcript thereof) by a nucleic acid amplification method using the primer or primer set according to the present invention, and detecting the amplification product is detected.
The detection of expression of the Nato3 gene by utilizing the nucleic acid amplification method can be performed, for example, by the following steps of:
(c) performing a nucleic acid amplification method by using a polynucleotide derived from a cell sample to be tested as a template and the polynucleotide primer or the polynucleotide primer set according to the present invention; and
(d) detecting a formed amplification product.
In step (c), mRNA prepared from the cell sample to be tested that is thought to contain dopaminergic neuron proliferative progenitor cells or complementary DNA (cDNA) transcribed from the mRNA can be used as the template.
The detection of the amplification product can be performed by using a nucleic acid amplification method such as a PCR method, a RT-PCR method, a real-time PCR method, or a LAMP method.
The cells in which the amplification product is detected are also expressing a Nato3 gene, and therefore can be determined as the dopaminergic neuron proliferative progenitor cells.
According to the third embodiment of the detection method according to the present invention, the antibody according to the present invention and the cell sample are contacted, and the antigen-antibody reaction is detected. Thus, the expression of a Nato3 gene can be detected in the cell sample.
The detection of expression of the Nato3 gene by utilizing the antigen-antibody reaction can be performed, for example, by the following steps of:
(e) contacting a protein derived from a cell sample to be tested, with the antibody according to the present invention; and
(f) measuring an antigen-antibody complex.
The method for detecting the antigen-antibody reaction is well-known for the skilled person, and, for example, a Nato3 protein can be detected in the cell sample to be tested that is thought to contain dopaminergic neuron proliferative progenitor cells by an immunological method. For the immunological method, a previously known method such as a immunohistologic staining method, an enzyme-linked immunosorbent assay, a western blotting method, an agglutination method, a competition method, or a sandwich method, can be applied to the cell sample subjected to appropriate treatment according to need, such as segregation or extraction operation of the cells. The immunohistologic staining method can be performed by, for example, a direct method by using a labeled antibody or an indirect method by using an labeled antibody against the antibody. For the labeling agent, a known labeling substance such as a fluorescent substance, a radioactive substance, an enzyme, a metal, or a pigment can be used.
The cells in which the antigen-antibody complex is detected are those expressing a Nato3 gene, and therefore can be determined as the dopaminergic neuron proliferative progenitor cells.
For use in the treatment of the Parkinson's disease, it is desirable that the purity of the dopaminergic neuron proliferative progenitor cells is high.
The accuracy of the detection or selection of the dopaminergic neuron proliferative progenitor cells can be enhanced by performing each of the above-described detection steps not only once but repeatedly.
Therefore, according to the detection method according to the present invention, the dopaminergic neuron proliferative progenitor cells can be detected or selected with high accuracy by performing the above-described step twice or more.
Moreover, the accuracy of detection or selection of the dopaminergic neuron proliferative progenitor cells can be enhanced by using together other marker genes, preferably dopaminergic neuron proliferative progenitor cell marker genes except for the Nato3 genes.
Therefore, according to the detection method according to the present invention, the dopaminergic neuron proliferative progenitor cells can be detected or selected with higher accuracy by using together dopaminergic neuron proliferative progenitor cell marker genes except for the Nato3 genes, postmitotic dopaminergic neuron precursor cell marker genes or the like, and detecting not only expression of the Nato3 gene but also expression of the other above-described marker genes
The dopaminergic neuron-related marker genes expressing selectively in each of the differentiation stages are shown in
In the detection method characterized in that the expression of the Nato3 gene is detected, the dopaminergic neuron proliferative progenitor cells can be detected or selected with high accuracy by detecting not only the Nato3 gene but also the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes by using together dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes.
Specifically, in step (a), step (c), or step (e), the dopaminergic neuron proliferative progenitor cells can be detected or selected with high accuracy by using the cells in which the expression of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes is detected as the cell sample to be tested. In this case, the cells in which the hybridization complex is detected in the step (b), the cells in which the amplification product is detected in the step (d), and the cells in which the antigen-antibody complex is detected in the step (f) each express a Nato3 gene, and the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes. Thus, the cells can be determined as the detected or selected dopaminergic neuron proliferative progenitor cells with high accuracy.
Moreover, the dopaminergic neuron proliferative progenitor cells can be detected or selected with high accuracy by performing step (g-1) of detecting expression of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes with respect to the cells in which the hybridization complex is detected in step (b), the cells in which the amplification product is detected in step (d), and the cells in which the antigen-antibody complex is detected in step (f), respectively. In this case, in step (g-1), the cells in which the expression of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes is detected are those expressing a Nato3 gene, and the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes. Thus, the cells can be determined as the detected or selected dopaminergic neuron proliferative progenitor cells with high accuracy.
In the detection method characterized in that the expression of the Nato3 gene is detected, by using together a postmitotic dopaminergic neuron precursor cell marker gene, it can be confirmed that the Nato3 gene is expressed but the expression of the postmitotic dopaminergic neuron precursor cell marker gene is not detected. Thus, the dopaminergic neuron proliferative precursor cells can be detected or selected with high accuracy.
Specifically, in step (a), step (c), or step (e), the dopaminergic neuron proliferative progenitor cells can be detected or selected with high accuracy by using the cells in which the expression of the postmitotic dopaminergic neuron precursor cell marker gene is not detected. In this case, the cells in which the hybridization complex is detected in step (b), the cells in which the amplification product is detected in step (d), and the cells in which the antigen-antibody complex is detected in step (f) each express a Nato3 gene but does not express the postmitotic dopaminergic neuron precursor cell marker gene. Thus, the cells can be determined as the detected or selected dopaminergic neuron proliferative progenitor cells with high accuracy.
Moreover, the dopaminergic neuron proliferative progenitor cells can be detected or selected with high accuracy by performing step (g-2) of detecting expression of the postmitotic dopaminergic neuron precursor cell marker gene with respect to the cells in which the hybridization complex is detected in step (b), the cells in which the amplification product is detected in step (d), and the cells in which the antigen-antibody complex is detected in step (f), respectively. In this case, in step (g-2), the cells in which the expression of the postmitotic dopaminergic neuron precursor cell marker gene is not detected are those expressing a Nato3 gene, but not the postmitotic dopaminergic neuron precursor cell marker gene. Thus, the cells can be determined as the detected or selected dopaminergic neuron proliferative progenitor cells with high accuracy.
“The dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes” includes a dopaminergic neuron proliferative progenitor cell marker gene except for a Nato3 gene which is expressed in the midbrain's most ventral ventricular region (VZ region), and includes an Lrp4 gene, an Msx1 gene, an Msx2 gene, and a Mash1 gene.
An Lrp4 gene is described in WO 2004/065599. A Mash1 gene is described in Kele J, Simplicio N, Ferri A L, Mira H, Guillemot F, Arenas E, Ang S L. Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons, and Development. 2006 February; 133(3):495-505. It has been confirmed by the present inventors that an Msx1 gene and an Msx2 gene are expressed selectively in the dopaminergic neuron proliferative progenitor cells (the data not shown).
The detection of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes is not limited as long as using a method by which expression of the known gene can be detected, and, for example, includes the above-described hybridization method, the nucleic acid amplification method, and the antigen-antibody reaction method.
“The postmitotic dopaminergic neuron precursor cell marker gene” includes a gene expressed in the midbrain's most ventral mantle layer (ML region), and includes a Nurr1 gene, an En1 gene, an En2 gene, a Ptx3 gene, and a TH gene. Moreover, the marker gene includes a gene expressed in the midbrain's most ventral ventricular region (VZ region), and includes a 65B13 gene.
A Nurr1 gene is described in Science. 1997 11; 276(5310):248-50. An En1 gene is described in J. Neurosci. 2001 21(9): 3126-34. An En2 gene is described in J. Neurosci. 2001 21(9) 3126-34. A Ptx3 gene is described in Proc. Natl. Acad. Sci. 1997 94: 13305-10. A TH gene is described in Science 1997 11; 276(5310):248-50. A 65B13 gene is described in WO 2004/038018.
The detection of the postmitotic dopaminergic neuron precursor cell marker gene is not particularly limited as long as using a method by which expression of the known gene can be detected, and, for example, includes the above-described hybridization method, the nucleic acid amplification method, and the antigen-antibody reaction method.
The present invention provides a detection kit for performing the detection method according to the present invention.
The first embodiment of the detection kit according to the present invention includes a detection kit for performing the detection method of the first embodiment according to the present invention, and specifically, a kit for detecting the expression of the Nato3 gene, including at least the probe according to the present invention. The probe may be labeled. The detection kit detects the expression of the Nato3 gene by a hybrid formation method.
Therefore, the detection method of the first embodiment can optionally further include various reagents for performing the hybrid formation method such as a substrate compound used for detection of the marker, hybridization buffer, instructions, equipment, and/or so forth.
For performing the detection with high accuracy, the detection kit of the first embodiment according to the present invention may further include the probe, the primer, the primer set, or the antibody which can detect the expression of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes, or the expression of the postmitotic dopaminergic neuron precursor cell marker gene. The probe, the primer, the primer set, or the antibody may be labeled. By any of the hybrid formation method, the nucleic acid amplification method, and the antigen-antibody reaction method, the detection kit further detects the expression of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes, or the expression of the postmitotic dopaminergic neuron precursor cell marker gene.
The second embodiment of the detection kit according to the present invention includes a detection kit for performing the detection method of the second embodiment according to the present invention, and specifically, a kit for detecting the expression of the Nato3 gene, including at least the primer according to the present invention or the primer set according to the present invention. The detection kit detects the expression of the Nato3 gene by the nucleic acid amplification method.
Therefore, the detection method of the second embodiment can optionally further include various reagents for performing the nucleic acid amplification method such as a buffer, an internal standard indicating that the PCR can normally progress, instructions, equipment, and/or so forth.
For performing the detection with high accuracy, the detection kit of the second embodiment according to the present invention may further include the probe, the primer, the primer set, or the antibody which can detect the expression of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes, or the expression of the postmitotic dopaminergic neuron precursor cell marker gene. The probe, the primer, the primer set, or the antibody may be labeled. By any of the hybrid formation method, the nucleic acid amplification method, and the antigen-antibody reaction method, the detection kit further detects the expression of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes, or the expression of the postmitotic dopaminergic neuron precursor cell marker gene.
The third embodiment of the detection kit according to the present invention includes a detection kit for performing the detection method of the third embodiment according to the present invention, and specifically, a kit for detecting the expression of the Nato3 gene, including at least the antibody according to the present invention. The antibody may be labeled. The detection kit detects the expression of the Nato3 gene by detecting the antigen-antibody reaction.
Therefore, the detection method of the third embodiment can optionally further include various reagents for performing the antigen-antibody reaction such as a secondary antibody used for the ELISA method or the like, a coloring reagent, a buffer, instructions, equipment, and/or so forth.
For performing the detection with high accuracy, the detection kit of the third embodiment according to the present invention may further include the probe, the primer, the primer set, or the antibody which can detect the expression of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes, or the expression of the postmitotic dopaminergic neuron precursor cell marker gene. The probe, the primer, the primer set, or the antibody may be labeled. By any of the hybrid formation method, the nucleic acid amplification method, and the antigen-antibody reaction method, the detection kit further detects the expression of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes, or the expression of the postmitotic dopaminergic neuron precursor cell marker gene.
The detection method according to the present invention can be applied to screening for effective substances for inducing differentiation into the dopaminergic neuron proliferative progenitor cells. Specifically, effective substances for inducing differentiation into dopaminergic neuron proliferative progenitor cells can be screened for by determining whether or not the addition of a substance to be tested has induced the differentiation into the dopaminergic neuron proliferative progenitor cells using expression of a Nato3 gene as an index.
Therefore, the present invention provides a method for screening for an effective substance for inducing differentiation into a dopaminergic neuron proliferative progenitor cell, comprising the following steps of:
(i) contacting a cell that can differentiate into a dopaminergic neuron proliferative progenitor cell, with a substance to be tested; and
(ii) detecting expression of a Nato3 gene in the cell contacted with the substance to be tested.
The cell that can differentiate into a dopaminergic neuron proliferative progenitor cell in step (i) can be preferably collected from an embryonic midbrain ventral region or from culture cells containing neuron progenitor cells induced to differentiate from ES cells.
“Contacting with a substance to be tested” in step (i) can be performed by, for example, adding the substance to be tested to culture cells containing the cells that can differentiate into a dopaminergic neuron proliferative progenitor cell.
“Substance to be tested” includes a synthesized low-molecular compound, a protein, a synthesized peptide, a purified or partially purified polypeptide, an antibody, a bacterium-releasing material (comprising bacterial metabolite), and a nucleic acid (such as antisense, ribozyme, and RNAi), and is preferably a synthesized low-molecular compound, but is not limited thereto. “Substance to be tested” may be a novel substance or a known substance.
In step (ii), according to the detection method of the present invention, the expression of the Nato3 gene can be detected.
Specifically, steps (a) and (b) are performed for the detection by utilizing the hybridization method. Steps (c) and (d) are performed for the detection by utilizing the nucleic acid amplification method. Steps (e) and (f) are performed for the detection by utilizing the antigen-antibody reaction. Thus, the expression of the Nato3 gene can be detected.
In step (ii), when the expression of the Nato3 gene is detected in the cell sample by contacting the substance to be tested, the substance can be determined as the effective substance for inducing differentiation into the dopaminergic neuron proliferative progenitor cells.
The substance specified by the screening method according to the present invention can be used as the effective substance for inducing differentiation into the dopaminergic neuron proliferative progenitor cells.
The present invention provides the method for screening for an effective substance for inducing differentiation into a dopaminergic neuron proliferative progenitor cell, further comprising the step of:
When the expression of the Nato3 gene is detected in step (ii) and the expression of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes is detected in step (iii), the substance can be determined as the effective substance for inducing differentiation into dopaminergic neuron proliferative progenitor cells, with high accuracy.
Step (iii) may be performed after step (i) and may be performed before or after the step (ii).
“The dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes” includes the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes that are expressed in the midbrain most ventral ventricular region (VZ region), and, for example, includes an Lrp4 gene, an Msx1 gene, an Msx2 gene, and a Mash1 gene.
The detection of the dopaminergic neuron proliferative progenitor cell marker gene except for the Nato3 genes is not particularly limited as long as using a method by which the expression of the known gene can be detected, for example, includes the above-described hybridization method, the nucleic acid amplification method, and the antigen-antibody reaction method.
The detection method according to the present invention can detect or select the dopaminergic neuron proliferative progenitor cells. The dopaminergic neuron proliferative progenitor cells for use in the treatment of the Parkinson's disease. Therefore, the dopaminergic neuron proliferative progenitor cells for use in the treatment of the Parkinson's disease can be produced from the detected or selected dopaminergic neuron proliferative progenitor cells using the expression of a Nato3 gene as an index.
The present invention provides a method for producing a dopaminergic neuron proliferative progenitor cell, comprising the steps of:
(iv) obtaining cells that can contain a dopaminergic neuron proliferative progenitor cell;
(v) detecting or selecting the dopaminergic neuron proliferative progenitor cell by using the detection method according to the present invention; and
(vi) culturing the cell detected or selected in step (v).
The present invention provides a method for treating the Parkinson's disease comprising the step of transplanting the dopaminergic neuron proliferative progenitor cells detected or selected by the detection method according to the present invention, or the dopaminergic neuron proliferative progenitor cells produced by the production method according to the present invention, into a mammal including a human.
According to the treatment method of the present invention, the transplanted dopaminergic neuron proliferative progenitor cells produce dopamine, and thus, the Parkinson's disease can be prevented and/or treated.
When the treatment method according to the present invention is performed, the cells that can contain the dopaminergic neuron proliferative progenitor cells can be collected from a mammal comprising a human, and preferably, the individual subjected to the transplantation or an aborted fetus.
The dopaminergic neuron proliferative progenitor cells can be transplanted into a brain, preferably, a midbrain.
In the present specification, “detection” includes “discrimination”.
Hereinafter, the present invention will be specifically explained by Examples, but the following Examples do not limit the scope of the present invention.
In order to confirm that a Nato3 gene is expressed in the cells of dopaminergic neuron lineage, expressions of mRNAs of Nato3, DAT, Lmx1a, and Lrp4 in each region of a mouse embryonic midbrain were investigated by a RT-PCR method according to the following protocol. Here, DAT is a marker gene of the dopaminergic neuron (Development. 2004; 131(5):1145-55.), Lmx1a is a marker gene of dopaminergic neurons and dopaminergic neuron progenitor cells (WO2005/052190), and Lrp4 is a marker gene of the dopaminergic neuron proliferative progenitor cells (WO2004/065599).
From a 12.5-day mouse (obtained from SLC) embryo, 4 regions (V region: most ventral region, VL region: ventral lateral region, DL region: dorsal lateral region, and D region: most dorsal region) of the midbrain shown in
Next, by using the cDNAs corresponding to the amplified cDNA of 4 ng, 0.4 ng, and 0.04 ng as templates, PCR was performed in the following reaction system.
After incubation for 2 minutes at 94° C., the amplification reaction for 30 seconds at 94° C., for 30 seconds at 65° C., and for 2 minutes at 72° C., was performed, and finally, incubation was performed for 2 minutes at 72° C. The PCR amplifications were performed at 26 cycles for Lrp4, DAT and Lmx1a, and at 25 cycles for Nato3.
The following primers were used in the PCR.
As a result, it became revealed that mRNA of Nato3 is selectively expressed in the V region of the midbrain in which the marker genes of dopaminergic neuron and dopaminergic neuron progenitor cells are expressed (
(2) Analysis by in situ Hybridization
Furthermore, in order to investigate the expression pattern in detail, by in situ hybridization according to the following protocol, expression analysis of mRNA of Nato3, Nurr1, and tyrosine hydroxylase (TH) was performed. Nurr1 and TH are marker genes that are known to be induced to express first after postmitotic in the dopaminergic neuron precursor cells (Science. 1997 11; 276(5310): 248-50.).
First, a DIG-probe was produced by the following method.
From a 12.5-day mouse (obtained from SLC) embryo, the midbrain afterbrain region was cut out, and the total RNA was prepared by using the RNeasy mini kit (Qiagen), and double-strand cDNA was synthesized by using the cDNA synthesis kit (TAKARA). By using the synthesized cDNA as a template, cDNAs of Nato3, Nurr1, and TH were amplified in the following reaction system.
The amplification was carried out under the conditions that incubation was performed for 5 minutes at 94° C., and then reactions for 30 seconds at 94° C., for 30 seconds at 65° C., and for 2 minutes at 72° C., were performed at 35 cycles, and finally, incubation was performed for 2 minutes at 72° C.
The following primers were used in the PCR.
The amplified cDNA fragments were cloned into pCRII (Invitrogen) and used as templates, and thereby, DIG-probes were synthesized by the following reaction system (all of the reagents were purchased from Roche)
After 2 hours at 37° C., DNaseI (Roche) treatment was performed for 15 minutes at 37° C., and the DIG-RNA probe was collected by ethanol precipitation.
Next, an 11.5-day mouse embryo was excised and fixed for 2 hours at 4° C. by using 4% PFA (WAKO)/PBS (−), and then, the solution was replaced at 4° C. overnight by 20% sucrose (WAKO)/PBS (−) and then the embryo was embedded with OCT (Sakura Seiki Co., Ltd.). Sections of 12 μm thickness were prepared and dried on slide glasses and then fixed again for 30 minutes at room temperature by using 4% PFA. After rinsing with PBS, hybridization (1 μg/ml DIG-RNA probe, 50% formamide (Nacalai Tesque, Inc.), 5×SSC, 1% SDS, 50 μg/ml yeast RNA (Sigma), 50 μg/ml heparin) was performed for 40 hours at 68° C. Then, rinsing (50% formamide, 5×SSC, and 1% SDS) was performed at 68° C. and further rinsing (50% formamide, 5×SSC) was performed at 68° C. After rinsing with 1×TBST at room temperature, blocking (blocking agent: Roche) was performed. Alkaline phosphatase-labeled anti-DIG antibody (DAKO) was reacted at 4° C. overnight, and after rinsing (1×TBST, 2 mM levamisole), NBT/BCIP (DAKO) was used as the substrate for coloring.
As a result, in the 11.5-day mouse embryo which is in the period of generating dopaminergic neurons, it became revealed that mRNA of Nato3 is strongly expressed in the V region in the same manner as TH and Nurr1 (
Next, by using anti-Nato3 antibody, the expression of Nato3 protein was studied. Moreover, double staining by using an anti-Lmx1a antibody and an anti-Nurr1 antibody was performed.
First, the 1-92 amino acid region of Nato3 protein was expressed as a fusion protein with a GST protein in E. Coli JM109 cell line (TAKARA) and collected. A rat was immunized with the collected fusion protein, and then, a lymphocyte cell was taken out and cell-fused to a myeloma P3U1, and thereby, an anti-Nato3 antibody-producing hybridoma was obtained (hybridoma production was outsourced to Kojin-Bio Co., Ltd.). Similarly, the 271-307 amino acid region of the Lmx1a protein was expressed as a fusion protein with the GST protein and collected. A hamster (obtained from SLC) was immunized with the collected fusion protein, and then, the lymphocyte cells were taken out and cell-fused to a myeloma (ATCC), and thereby, an anti-Lmx1a antibody-producing hybridoma was obtained.
A 11.5-day mouse embryo was excised and fixed for 2 hours at 4° C. by using 4% PFA (WAKO)/PBS (−), and then, the solution was replaced at 4° C. overnight by 20% sucrose (WAKO)/PBS (−) and then the embryo was embedded with OCT (Sakura Seiki Co., Ltd.). Sections of 12 μm thickness were prepared, mounted on slide glasses, dried for 30 minutes at room temperature, and then moistened again with PBS (−). Next, blocking (Blockase (Dainippon Sumitomo Pharma Co., Ltd.)) was performed for 30 minutes at room temperature, and then, reaction with a primary antibody (anti-Nato3 and anti-Lmx1a antibodies from above-described hybridoma culture supernatant, anti-Nurr1 antibody from Santa Cruz) was performed for one hour at room temperature, and then, reaction was further performed at 4° C. overnight. By using 0.1% Tween-20/PBS (−), rinsing was performed for 15 minutes at room temperature three times. Next, a fluorescence-labeled secondary antibody (Jackson) was reacted for one hour at room temperature and rinsing was performed in the same manner, and then, rinsing with PBS(−) was performed for 10 minutes at room temperature, and sealed.
As a result, it became revealed that Nato3 is expressed in the midbrain of the 11.5-day mouse embryo as protein as well as mRNA (
Whether a Nato3 gene is expressed when ES cells are induced to differentiate into dopaminergic neurons was studied.
First, according to the SDIA method (Kawasaki et al. Neuron. 2000 28(1):31-40.), ES cells (mouse CCE strain provided from Mr. Nishikawa in Riken CDB, Kawasaki et al. Neuron. 2000 28(1):31-40.) was induced to differentiate into dopaminergic neurons. The cells were collected after 4, 6, 8, 10, 12 days after the induction. The total RNA was prepared by using the RNeasy mini kit (Qiagen), and RT-PCR was performed. First, with respect to 1 μg of the total RNA, cDNA synthesis was performed by using the RNA PCR kit (TAKARA). By using the cDNAs corresponding to 10 ng, 1 ng, and 0.1 ng as templates, PCR was performed in the following reaction system.
After incubation for 2 minutes at 94° C., the reaction for 30 seconds at 94° C., for 30 seconds at 65° C., and for 2 minutes at 72° C., was performed at 35 cycles, and finally, incubation was performed for 2 minutes at 72° C.
The following primers were used in the PCR.
In addition, for Nato3 and Lmx1a, the primers of Example 1 were used. Moreover, Ptx3 and En1 are known as markers of the postmitotic dopaminergic neuron precursor cells.
As a result, expression of mRNA of Nato3 was not recognized in ES cells (CCE) and stromal cells (PA6), but it became revealed that as a result of the differentiation induction, the expression is induced from the fourth day in the same manner as Lmx1a, which is a marker gene of the dopaminergic neuron proliferative progenitor cells (
In order to confirm that a Nato3 gene is expressed in the dopaminergic neuron proliferative progenitor cells, the dopaminergic neuron proliferative progenitor cells were separated from the cells derived from the midbrain of a 12.5-day mouse embryo and SDIA differentiation induction cells respectively using a Lrp4 gene which is expressed selectively in the dopaminergic neuron proliferative progenitor cells as a marker, and the expression of mRNA of Nato3 in these cells was investigated.
First, a gene sequence encoding the extracellular region (161-502 amino acids) in an Lrp4 gene was gene-transfected into 293E cells (ATCC), and the extracellular region of the Lrp4 protein was expressed and collected. A hamster (obtained from SLC) was immunized with the collected protein, and then, lymphocytic cells were extracted and cell-fused with myeloma cells (ATCC) to obtain an anti-Lrp4 antibody-producing hybridoma.
The group of the cells containing the dopaminergic neuron progenitor cells derived from the midbrain of the 12.5-day mouse embryo or induced to differentiate from ES cells in vitro was dispersed by using a cell dissociation Buffer™ (Invitrogen), and then, without being subjected to fixation and permeabilization treatments, the cells were stained for 20 minutes at 4° C. by using an anti-Lrp4 monoclonal antibody (a culture supernatant diluted to 1/2, 1% fetal bovine serum (JRH), 1 mM EDTA/SDIA differentiation medium (Kawasaki et al. Neuron (2000)28(1):31-40)). Then, with 1% fetal bovine serum and 1 mM EDTA/SDIA differentiation medium, rinsing was performed for 3 minutes at 4° C. three times, and the cells were stained for 20 minutes at 4° C. by using a biotin-labeled anti-hamster IgG antibody (Jackson, 10 μg/ml, 1% fetal bovine serum, 1 mM EDTA/SDIA differentiation medium), and then, rinsing was performed in the same manner. The cells were stained for 20 minutes at 4° C. by using a PE-labeled streptavidin (Pharmingen, 20 μg/ml, 1% fetal bovine serum, 1 mM EDTA/SDIA differentiation medium), and then, rinsing was performed in the same manner. After the staining, Lrp4 positive cells and negative cells were separated by a cell sorter (FACS vantage SE, Becton Dickinson) (
Next, by using the cDNAs corresponding to the amplified cDNA of 4 ng, 0.4 ng, and 0.04 ng as templates, PCR was performed in the following reaction system.
The primers having the above-described sequences were used.
After incubation for 2 minutes at 94° C., the amplification reaction for 30 seconds at 94° C., for 30 seconds at 65° C., and for 2 minutes at 72° C., was performed, and finally, incubation was performed for 2 minutes at 72° C. The amplifications of PCR were performed at 24 cycles for Lmx1a and Nurr1 and at 26 cycles for the other.
As a result, in the cells derived from the midbrain of the 12.5-day mouse embryo, mRNA of Nato3 was strongly expressed in a Lrp4-positive cell population (namely, dopaminergic neuron proliferative progenitor cells) (
Therefore, it was revealed that a Nato3 gene is a useful marker for discriminating not only the dopaminergic neuron proliferative progenitor cells derived from the embryonic midbrain but also the dopaminergic neuron proliferative progenitor cells induced to differentiate from ES cells in vitro.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-237759 | Aug 2005 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2006/316249 | 8/18/2006 | WO | 00 | 2/15/2008 |
