# Patent

## Title
Genetically modified animal for use in evaluating harmfulness of test substance

## Abstract

A genetically modified animal that includes an introduced DNA including a functionally linked aryl hydrocarbon receptor-binding enhancer located 5′-upstream of a tyrosine hydroxylase gene, promoter of any type, reporter gene, and poly(A) addition signal.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2008-244392, filed Sep. 24, 2008, the entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a genetically modified animal for use in evaluating the harmfulness of test substances and a method for evaluating the action of test substances in the genetically modified animal or part thereof.
2. Description of the Related Art
There have been concerns about the effect of environmental pollutant chemicals such as dioxins, PCBs, brominated flame retardants, and some agricultural chemicals on human health. In addition, many of various chemicals used in industrial activity or everyday life may have an unknown effect on human health.
Such chemicals are considered to act on biological functions such as liver function, reproductive function, immune function, and neural function and to have a harmful effect on human health.
Therefore, techniques for detecting and/or evaluating a biological function on which a specific chemical substance can act are very important for definition of handling or control of chemicals or for treatment of a human exposed to chemicals. Particularly, in recent years, it has been pointed out that exposure to some chemicals during the fetal or infant period could cause infants to have autism or higher brain dysfunction such as hyperactivity disorder. Therefore, there has been a demand for the development of techniques to evaluate the effect of chemicals on the neural function.
Currently, techniques to evaluate the effect of chemicals on biological functions are broadly classified into in vitro evaluation techniques using single cells and in vivo evaluation techniques using animal individuals.
The in vitro evaluation techniques include a technique of using mouse liver-derived Hepalc1c7 cells to evaluate the effect of test substances on the liver function (U.S. Pat. No. 5,854,010) and a method of using human mammary gland-derived MCF-7 cells to evaluate the effect of test substances on the reproductive function (JP-A 2002-253231). As regards the neural function, an in vitro evaluation method using mouse Neuro2a neuroblastoma cells has already been reported by the present applicant. This method includes transfecting the cells with a reporter gene under the control of a promoter having an aryl hydrocarbon receptor-binding enhancer sequence for a tyrosine hydroxylase gene (TH) gene and a TH gene core promoter sequence to produce genetically modified cells and evaluating the effect of a test substance on the central nervous system in the genetically modified cells. Concerning the in vivo evaluation techniques, there have been reported methods for evaluating the effect of test substances on the reproductive function, which use genetically modified mice produced by the introduction of a reporter gene under the control of an Mvh gene or c-mos gene promoter (JP-A 2001-8577 and 2001-8578).
BRIEF SUMMARY OF THE INVENTION
In view of the above circumstances, an object of the invention is to provide a genetically modified animal for use in evaluating the harmfulness or effect of test substances on a living body and a method for evaluating the effect of test substances in the animal or part thereof. A specific object of the invention is to provide a genetically modified animal for use in evaluating the effect of test substances on the neural function and a method of using the genetically modified animal or part thereof to evaluate the effect of test substances on the neural function.
To achieve the object, the invention provides:
(1) a genetically modified animal including introduced DNA in which an aryl hydrocarbon receptor-binding enhancer located 5′-upstream of a tyrosine hydroxylase gene, a promoter of any type, a reporter gene, and a poly(A) addition signal are functionally linked; and
(2) a method for determining the harmfulness of a test substance, including bringing the test substance into contact with the genetically modified animal of item (1) or part thereof and detecting the expression of the reporter gene.
The invention provides a genetically modified animal for use in evaluating the harmfulness or effect of a test substance on a living body and a method of evaluating the effect of a test substance in the animal or part thereof. Specifically, the invention provides a genetically modified animal for use in evaluating the effect of a test substance on a neural function and a method of using the genetically modified animal or part thereof to evaluate the effect of a test substance on a neural function.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention, and together with the general description given above and the detailed description of the embodiments given below, serve to explain the principles of the invention.
FIG. 1 is a diagram showing a gene transcription activating mechanism;
FIG. 2 is a diagram showing a vector preparation flow;
FIG. 3 is a diagram showing DNA to be introduced into a fertilized egg;
FIG. 4 shows the result of Southern hybridization indicating introduced DNA-derived signals;
FIG. 5 shows the result of electrophoresis indicating introduced DNA-derived signals;
FIG. 6 shows luciferase luminescence in genetically modified mice;
FIG. 7 shows an increase in luminescence in the vicinity of mouse midbrain or hippocampus;
FIG. 8 shows cultured nerve cells; and
FIG. 9 is a graph showing a TCDD concentration-dependent increase in the degree of luminescence.



DETAILED DESCRIPTION OF THE INVENTION
At present, there has been reported an in vitro evaluation technique of using mouse Neuro2a neuroblastoma cells to evaluate the effect of test substances on the neural function. In such an evaluation technique using single cells, however, it is difficult to precisely evaluate the effect of test substances at the individual animal level. For example, this is considered to be because there is a high possibility that single cells will not necessarily reproduce the uptake or distribution of test substances or the metabolism of test substances in the same manner as in an animal individual. Therefore, it is considered that when the effect of test substances on biological functions is evaluated, an in vitro evaluation technique should preferably be used in combination with an in vivo evaluation technique. Thus, the inventors have noted that conventionally, in vivo evaluation techniques effective for evaluating neural functions have not yet been established, and have worked on the development of in vivo evaluation techniques to evaluate the effect of test substances on neural functions.
The TH gene is a gene encoding a tyrosine hydroxylase which is a rate-limiting enzyme of dopamine biosynthesis, and it is known that the modification of the expression of the gene by exposure to chemicals can affect neural functions. The inventors have made investigations on in vivo techniques of using the expression of the TH gene as an indicator to evaluate the effect of chemicals on neural functions. As a result, the inventors have made a genetically modified mouse having an introduced DNA in which an aryl hydrocarbon receptor (hereinafter also referred to as “AhR”)-binding enhancer of the TH gene, any promoter, a reporter gene, and a poly(A) addition signal are linked together.
The genetically modified mouse of the invention expresses the reporter gene in the central nervous system, and the expression correlates with the expression of the TH gene. Therefore, the use of the genetically modified mouse allows easy and fast evaluation of the effect of test substances on the expression of the TH gene, namely, the effect of test substances on the central nervous system, by in vivo biochemical measurement using the expression of the reporter gene as an indicator.
[Genetically Modified Mouse]
The invention will be described in detail below. The genetically modified mouse of the invention may be obtained by transfecting a fertilized egg or an early embryo with DNA in which an AhR-binding enhancer for a TH gene, a promoter of any type, a reporter gene, and a poly(A) addition signal are functionally linked.
As used herein, the term “functionally linked” means that the regions are linked in such a way that each region can perform its function. For example, a functionally linked promoter or reporter gene is a promoter or reporter gene linked in such a way that the promoter can be active or the reporter gene expression can be enhanced in the genetically modified animal according to the invention.
In an embodiment of the invention, the DNA to be introduced into a mouse has an AhR-binding enhancer, any promoter, and a reporter gene.
[Enhancer]
As used herein, the term “enhancer” refers to a specific nucleotide sequence that exists on a genome, can bind a transcription factor activated by a substrate, and has the function of activating the transcription of a downstream gene upon binding of the transcription factor. The AhR-binding enhancer is a nucleotide sequence that can bind AhR (a receptor-type transcription factor) activated by binding of dioxin and has the function of activating the transcription of the downstream gene upon the binding of AhR.
In an embodiment of the invention, the AhR-binding enhancer should include the nucleotide sequence represented by SEQ ID NO:1 as the minimal sequence. Those of skill in the art may select other AhR-binding enhancers from those known in the art or from those that comprise SEQ ID NO: 1, which are at least 80%, 90%, 95%, or 96.8% identical to SEQ ID NO: 1, or which differ from SEQ ID NO: 1 by the deletion, substitution or addition of 1, 2, 3, 4, 5 bases.







[SEQ ID NO: 1]









TH gene AHR-binding enhancer (1)










[Minimal sequence]




GTCTTCATGT CGTGTCTAGG GCGG
(SEQ ID NO: 1)






This nucleotide sequence is derived from a region (−214 by to −237 bp) 5′-upstream of a mouse TH gene. In an embodiment of the invention, a nucleotide sequence in a wider region 5′-upstream of the TH gene, such as the nucleotide sequence represented by SEQ ID NO:2 (−175 by to −237 bp), may be used as the AhR-binding enhancer, as long as it includes the nucleotide sequence represented by SEQ ID NO:1.







[SEQ ID NO: 2]









TH gene AHR-binding enhancer (2)



[63 bp sequence]







(SEQ ID NO: 2)









GTCTTCATGT CGTGTCTAGG GCGGAGGGTG ATTCAGAGGC


 



AGGTGCCTGC GACAGTGGAT GCA


 



(Underline: SEQ ID NO: 1 = AHR-binding enhancer



(1))






Two or more AhR-binding enhancers may also be linked and used together, and the AhR-binding enhancer may be used in an orientation the same as the orientation on the mouse genome (forward) or in an orientation different from the orientation on the mouse genome (reverse). When two or more AhR-binding enhancers are linked and used together, a linking moiety including a linker sequence of about 1 to about 50 bases may be interposed between the enhancers, as long as the function of the enhancer is conserved. In an embodiment of the invention, another example of the AhR-binding enhancer may be the nucleotide sequence represented by SEQ ID NO:3.







[SEQ ID NO: 3]


TH gene AHR-binding enhancer (3)


[SEQ ID NO: 2 x6, in the orientational order of


reverse, reverse, forward, forward, forward, and


forward]


(SEQ ID NO: 3)


TAGATCTAAT TGCATCCACT TCGCAGGCAC CTCCTCTGAA


 


TCACCCTCCG CCCTAGACAC GACATGAAGA CTGCATCCAC


 


TGTCGCAGGC ACCTGCCTCT GAATCACCCT CCGCCCTAGA


 


CACGACATGA AGACAGGGGC TGGCGCCAGC CCCTGTCTTC


 


ATGTCGTGTC TAGGGCGGAG GGTGATTCAG AGGCAGGTGC


 


CTGCGACAGT GGATGCAGTC TTCATGTCGT GTCTAGGGCG


 


GAGGGTGATT CAGAGGAGGT GCCTGCGACA GTGGATGCAA


 


TTAGATCTAG CCAGCCCCTG TCTTCATGTC GTGTCTAGGG


 


CGGAGGGTGA TTCAGAGGCA GGTGCCTGCG ACAGTGGATG


 


CAGTCTTCAT GTCGTGTCTA GGGCGGAGGG TGATTCAGAG


 


GCAGGTGCCT GCGACAGTGG ATGCAATTAG ATCTA


 


(Underline: SEQ ID NO: 2 = AHR-binding enhancer


(2) x1)






This enhancer includes six SEQ ID NO: 2 nucleotide sequences linked together in the orientational order of reverse, reverse, forward, forward, forward, and forward from the 5′ side. This enhancer may be prepared by a combination of known genetic manipulation methods including polymerase chain reaction (PCR) and so on.
The enhancer may include one or more SEQ ID NO: 1 or NO: 2 nucleotide sequences, typically one, two, three, four, five, six, seven, eight, nine, ten, or more SEQ ID NO: 1 or NO: 2 nucleotide sequences, preferably two to eight SEQ ID NO: 1 or NO: 2 nucleotide sequences, most preferably three to six SEQ ID NO: 1 or NO: 2 nucleotide sequences. As described above, the orientation of the SEQ ID NO: 1 or NO: 2 sequence in the enhancer may be the same as the orientation of the enhancer sequence 5′-upstream of the mouse TH gene with respect to the TH structural gene (namely, the forward orientation), different from that (namely, the reverse orientation), or a combination thereof.
In an embodiment of the invention, the enhancer region to be used may be any other region than SEQ ID NOs: 1, 2 and 3, as long as it can enhance the activity of the transcription of the downstream gene in response to the test substance in the transcriptional regulatory region of the TH gene. For example, the transcriptional regulatory region of the TH gene is the region (2.5 kb) shown in SEQ ID NO: 17. The transcriptional regulatory region of the TH gene may be a region (2,500 bp) 5′-upstream of the TH gene in any animal species, such as a region (2,500 bp) 5′-upstream of the human, mouse, or rat TH gene.
The enhancer region capable of enhancing the activity of the transcription of the downstream gene in response to the test substance may also contain a region capable of enhancing the promoter activity.
[Promoter]
The promoter is a nucleotide sequence that includes an RNA polymerase-binding sequence and is essential to initiate the transcription of a gene. In an embodiment of the invention, the promoter is preferably the TH gene promoter shown in SEQ ID NO:4.







[SEQ ID NO: 4]









TH gene promoter







(SEQ ID NO: 4)









GTGGGGGACC CAGAGGGGCT TTGACGTCAG CCTGGCCTTT


 



AAGAGGCCGC CTGCCTGGCA AGGGCTGTGG AGACAGAACT


 



CGGGACCACC AGCTT






The promoter to be used is not limited thereto and may be any other known promoter capable of being active in a mammal, such as a simian virus 40 (SV40) early or late promoter, a human herpes virus 1 thymidine kinase promoter, or a cytomegalovirus promoter.
The promoter may be obtained by a combination of known genetic manipulation methods including polymerase chain reaction (PCR) and so on. The resulting promoter may be ligated to the 3′-end of the enhancer with a DNA ligase and so on. When the enhancer and the promoter are linked to each other, a linking moiety including a linker sequence of about 1 to 50 bases may be interposed therebetween, as long as the function of each of the enhancer and the promoter is conserved.
In an embodiment of the invention, an example of the nucleotide sequence including the enhancer and the promoter that are functionally linked together includes the nucleotide sequence shown in SEQ ID NO: 5.







[SEQ ID NO: 5]









AhR-binding enhancer (3) + TH gene promoter



[SEQ ID NO: 3 + SEQ ID NO: 4]







(SEQ ID NO: 5)









TAGATCTAAT TGCATCCACT TCGCAGGCAC CTCCTCTGAA


 



TCACCCTCCG CCCTAGACAC GACATGAAGA CTGCATCCAC


 



TGTCGCAGGC ACCTGCCTCT GAATCACCCT CCGCCCTAGA


 



CACGACATGA AGACAGGGGC TGGCGCCAGC CCCTGTCTTC


 



ATGTCGTGTC TAGGGCGGAG GGTGATTCAG AGGCAGGTGC


 



CTGCGACAGT GGATGCAGTC TTCATGTCGT GTCTAGGGCG


 



GAGGGTGATT CAGAGGAGGT GCCTGCGACA GTGGATGCAA


 



TTAGATCTAG CCAGCCCCTG TCTTCATGTC GTGTCTAGGG


 



CGGAGGGTGA TTCAGAGGCA GGTGCCTGCG ACAGTGGATG


 



CAGTCTTCAT GTCGTGTCTA GGGCGGAGGG TGATTCAGAG


 



GCAGGTGCCT GCGACAGTGG ATGCAATTAG ATCTAGGGCT


 



CGAGGTGGGG GACCCAGAGG GGCTTTGACG TCAGCCTGGC


 



CTTTAAGAGG CCGCCTGCCT GGCAAGGGCT GTGGAGACAG


 



AACTCGGGAC CACCAGCTT


 



(Underline: SEQ ID NO: 4 = TH gene promoter)






The nucleotide sequence represented by SEQ ID NO: 5 includes the enhancer shown in SEQ ID NO: 3 and the TH gene promoter shown in SEQ ID NO: 4 that are linked with a linker sequence interposed therebetween.
[Reporter Gene]
In an embodiment of the invention, the reporter gene is preferably a luciferase gene encoding an enzyme which catalyzes bioluminescence reaction in firefly, in view of enzyme reaction rate and reaction quantification. However, the reporter gene to be used is not limited thereto and may be any other known reporter gene such as a green fluorescent protein gene, a p-galactosidase gene, or a chloramphenicol acetyltransferase gene.
The reporter gene to be used may have the original nucleotide sequence of a naturally-occurring gene or may be a gene modified by mutagenesis methods such as site-directed mutagenesis. Alternatively, the reporter gene to be used may have a modified nucleotide sequence to express a fusion protein with another protein or polypeptide, as long as the function of the protein encoded by the reporter gene is not lost.
A commercially available gene may be purchased and used as the reporter gene, or the reporter gene may be prepared by a combination of known genetic manipulation methods including polymerase chain reaction (PCR) and so on. In the case of the luciferase gene, for example, the firefly luciferase gene shown in SEQ ID NO:7 may be used, which is incorporated in PGV vector series (Toyo B-Net Co., Ltd.).







[SEQ ID NO: 7]









Luciferase gene







(SEQ ID NO: 7)









ATGGAAGACG CCAAAAACAT AAAGAAAGG CCCGGCGCCA


 



TTCTATCCGC TGGAAGATGG AAAGATGGAA CCGCTGGAG


 



AGCAACTGCA TAAGGCTATG AAGAGATACG CCCTGGTTCC


 



TGGAACAAT TGCTTTTACA GATGCACATA TCGAGGTGGA


 



CATCACTTAC GCTGAGTAC TTCGAAATGT CCGTTCGGTT


 



GGCAGAAGCT ATGAAACGAT ATGGGCTGA ATACAAATCA


 



CAGAATCGTC GTATGCAGTG AAAACTCTCT TCAATTCTT


 



TATGCCGGTG TTGGGCGCGT TATTTATCGG AGTTGCAGTT


 



GCGCCCGCGA ACGACATTTA TAATGAACGT GAATTGCTCA


 



ACAGTATGGG CATTTCGCAG CCTACCGTGG TGTTCGTTTC


 



CAAAAAGGGG TTGCAAAAAA TTTTGAACGT GCAAAAAAAG


 



CTCCCAATCA TCCAAAAAAT TATTATCATG GATTCTAAAA


 



CGGATTACCA GGGATTTCAG TCGATGTACA CGTTCGTCAC


 



ATCTCATCTA CCTCCCGGTT TTAATGAATA CGATTTTGTG


 



CCAGAGTCCT TCGATAGGGA CAAGACAATT GCACTGATCA


 



TGAACTCCTC TGGATCTACT GGTCTGCCTA AAGGTGTCGC


 



TCTGCCTCAT AGAACTGCCT GCGTGAGATT CTCGCATGCC


 



AGAGATCCTA TTTTTGGCAA TCAAATCATT CCGGATACTG


 



CGATTTTAAG TGTTGTTCCA TTCCATCACG GTTTTGGAAT


 



GTTTACTACA CTCGGATATT TGATATGTGG ATTTCGAGTC


 



GTCTTAATGT ATAGATTTGA AGAAGAGCTG TTTCTGAGGA


 



GCCTTCAGGA TTACAAGATT CAAAGTGCGC TGCTGGTGCC


 



AACCCTATTC TCCTTCTTCG CCAAAAGCAC TCTGATTGAC


 



AAATACGATT TATCTAATTT ACACGAAATT GCTTCTGGTG


 



GCGCTCCCCT CTCTAAGGAA GTCGGGGAAG CGGTTGCCAA


 



GAGGTTCCAT CTGCCAGGTA TCAGGCAAGG ATATGGGCTC


 



ACTGAGACTA CATCAGCTAT TCTGATTACA CCCGAGGGGG


 



ATGATAAACC GGGCGCGGTC GGTAAAGTTG TTCCATTTTT


 



TGAAGCGAAG GTTGTGGATC TGGATACCGG GAAAACGCTG


 



GGCGTTAATC AAAGAGGCGA ACTGTGTGTG AGAGGTCCTA


 



TGATTATGTC CGGTTATGTA AACAATCCGG AAGCGACCAA


 



CGCCTTGATT GACAAGGATG GATGGCTACA TTCTGGAGAC


 



ATAGCTTACT GGGACGAAGA CGAACACTTC TTCATCGTTG


 



ACCGCCTGAA GTCTCTGATT AAGTACAAAG GCTATCAGGT


 



GGCTCCCGCT GAATTGGAAT CCATCTTGCT CCAACACCCC


 



AACATCTTCG ACGCAGGTGT CGCAGGTCTT CCCGACGATG


 



ACGCCGGTGA ACTTCCCGCC GCCGTTGTTG TTTTGGAGCA


 



CGGAAAGACG ATGACGGAAA AAGAGATCGT GGATTACGTC


 



GCCAGTCAAG TAACAACCGC GAAAAAGTTG CGCGGAGGAG


 



TTGTGTTTGT GGACGAAGTA CCGAAAGGTC TTACCGGAAA


 



ACTCGACGCA AGAAAAATCA GAGAGATCCT CATAAAGGCC


 



AAGAAGGGCG GAAAGATCGC CGTGTAA






When the luciferase gene of this vector is used for a fusion gene, DNA encoding a protein or peptide to be fused may be inserted in frame into the NcoI site incorporated on the initiation codon of the luciferase gene. In order to prepare a fusion gene encoding a luciferase protein fused with a c-myc tag sequence at the N-terminus, for example, the nucleotide sequences represented by SEQ ID NO: 8 and SEQ ID NO: 9 may be annealed to each other and then inserted between the Hind III and Nco I sites of the vector PGV-P2, so that the c-myc-tagged luciferase fusion gene shown in SEQ ID NO: 10 can be prepared.







[SEQ ID NO: 8]









c-myc tag (forward)







(SEQ ID NO: 8)









AGCTTATCAT GGAGCAGAAA CTCATCTCTG AAGAAGATCT


 



GGCCCGGGCG TC


 



(Underline: c-myc tag coding sequence)


 







[SEQ ID NO: 9]









c-myc tag (reverse)







(SEQ ID NO: 9)









GATGGACGCC CGGGCCAGAT CTTCTTCAGA GATGAGTTTC


 



TGCTCCATGA TA


 







[SEQ ID NO: 10]









c-myc tag-luciferase fusion gene







(SEQ ID NO: 10)









ATGGAGCAGA AACTCATCTC TGAAGAAGAT CTGGCCCGGG


 



CGTCCATGGA AGACGCCAAA AACATAAAGA AAGGCCCGGC


 



GCCATTCTAT CCGCTGGAAG ATGGAAAGAT GGAACCGCTG


 



GAGAGCAACT GCATAAGGCT ATGAAGAGAT ACGCCCTGGT


 



TCCTGGAACA ATTGCTTTTA CAGATGCACA TATCGAGGTG


 



GACATCACTT ACGCTGAGTA CTTCGAAATG TCCGTTCGGT


 



TGGCAGAAGC TATGAAACGA TATGGGCTGA ATACAAATCA


 



CAGAATCGTC GTATGCAGTG AAAACTCTCT TCAATTCTTT


 



ATGCCGGTGT TGGGCGCGTT ATTTATCGGA GTTGCAGTTG


 



CGCCCGCGAA CGACATTTAT AATGAACGTG AATTGCTCAA


 



CAGTATGGGC ATTTCGCAGC CTACCGTGGT GTTCGTTTCC


 



AAAAAGGGGT TGCAAAAAAT TTTGAACGTG CAAAAAAAGC


 



TCCCAATCAT CCAAAAAATT ATTATCATGG ATTCTAAAAC


 



GGATTACCAG GGATTTCAGT CGATGTACAC GTTCGTCACA


 



TCTCATCTAC CTCCCGGTTT TAATGAATAC GATTTTGTGC


 



CAGAGTCCTT CGATAGGGAC AAGACAATTG CACTGATCAT


 



GAACTCCTCT GGATCTACTG GTCTGCCTAA AGGTGTCGCT


 



CTGCCTCATA GAACTGCCTG CGTGAGATTC TCGCATGCCA


 



GAGATCCTAT TTTTGGCAAT CAAATCATTC CGGATACTGC


 



GATTTTAAGT GTTGTTCCAT TCCATCACGG TTTTGGAATG


 



TTTACTACAC TCGGATATTT GATATGTGGA TTTCGAGTCG


 



TCTTAATGTA TAGATTTGAA GAAGAGCTGT TTCTGAGGAG


 



CCTTCAGGAT TACAAGATTC AAAGTGCGCT GCTGGTGCCA


 



ACCCTATTCT CCTTCTTCGC CAAAAGCACT CTGATTGACA


 



AATACGATTT ATCTAATTTA CACGAAATTG CTTCTGGTGG


 



CGCTCCCCTC TCTAAGGAAG TCGGGGAAGC GGTTGCCAAG


 



AGGTTCCATC TGCCAGGTAT CAGGCAAGGA TATGGGCTCA


 



CTGAGACTAC ATCAGCTATT CTGATTACAC CCGAGGGGGA


 



TGATAAACCG GGCGCGGTCG GTAAAGTTGT TCCATTTTTT


 



GAAGCGAAGG TTGTGGATCT GGATACCGGG AAAACGCTGG


 



GCGTTAATCA AAGAGGCGAA CTGTGTGTGA GAGGTCCTAT


 



GATTATGTCC GGTTATGTAA ACAATCCGGA AGCGACCAAC


 



GCCTTGATTG ACAAGGATGG ATGGCTACAT TCTGGAGACA


 



TAGCTTACTG GGACGAAGAC GAACACTTCT TCATCGTTGA


 



CCGCCTGAAG TCTCTGATTA AGTACAAAGG CTATCAGGTG


 



GCTCCCGCTG AATTGGAATC CATCTTGCTC CAACACCCCA


 



ACATCTTCGA CGCAGGTGTC GCAGGTCTTC CCGACGATGA


 



CGCCGGTGAA CTTCCCGCCG CCGTTGTTGT TTTGGAGCAC


 



GGAAAGACGA TGACGGAAAA AGAGATCGTG GATTACGTCG


 



CCAGTCAAGT AACAACCGCG AAAAAGTTGC GCGGAGGAGT


 



TGTGTTTGTG GACGAAGTAC CGAAAGGTCT TACCGGAAAA


 



CTCGACGCAA GAAAAATCAG AGAGATCCTC ATAAAGGCCA


 



AGAAGGGCGG AAAGATCGCC GTGTAA


 



(Underline: part of SEQ ID NO: 8 = c-myc tag



coding sequence)






The reporter protein produced from such a fusion gene can be detected with a tag-specific antibody and therefore is effective for detecting a certain reporter protein to which no effective antibody is available. The reporter gene prepared as described above may be ligated to the nucleotide sequence including the functionally linked enhancer and promoter, for example, the 3′-end of the nucleotide sequence represented by SEQ ID NO: 5, using a DNA ligase and so on. When the promoter and the reporter are linked to each other, a linking moiety including a linker sequence of about 1 to 50 bases may be interposed therebetween, as long as the function of each of the promoter and reporter genes is conserved.
[Poly(A) Addition Signal]
In an embodiment of the invention, the poly(A) addition signal to be used may be any known transcription termination signal sequence capable of functioning to terminate the transcription of a mammal gene. Examples include an SV40 virus late poly(A) addition signal and a bovine growth hormone gene poly(A) addition signal. An example of the SV40 virus late poly(A) addition signal nucleotide sequence is shown in SEQ ID NO: 11.







[SEQ ID NO: 11]









SV40 virus late poly (A) addition signal







(SEQ ID NO: 11)









AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG


 



CAGTGAAAAA AATGCTTTAT TTGTGAAATT TGTGATGCTA


 



TTGCTTTATT TGTAACCATT ATAAGCTGCA ATAAACAAGT


 



TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG


 



GGGGAGGTGT GGGAGGTTTT TTAAAGCAAG TAAAACCTCT


 



ACAAATGTGG TA






However, the SV40 virus late poly(A) addition signal that may be used to carry out the invention is not limited thereto, and modifications of the sequence may also be used, as long as they do not lose the function of serving as a poly(A) addition signal.
The poly(A) addition signal to be used may be linked to the 3′-end of the reporter gene. The reporter gene and the poly(A) addition signal may be ligated using a DNA ligase or the like, or a commercially available vector including the reporter gene and the poly(A) addition signal that are already linked together may be used without modification. A linking moiety including a linker sequence of about 1 to 50 bases may be interposed between the reporter gene and the poly(A) addition signal, as long as the function of each of the reporter gene and the poly(A) addition signal is conserved.
[Introduced DNA]
In an aspect of the invention, DNA including the functionally linked enhancer, promoter, reporter gene, and poly(A) addition signal is introduced into a mouse so that a genetically modified mouse is produced. An example of the DNA to be introduced into a mouse is shown in SEQ ID NO: 6.








[SEQ ID NO: 6]



AhR-binding enhancer (3) + TH gene promoter +



c-myc tag-luciferase gene + SV40 virus late poly


(A) signal [SEQ ID NO: 3 + SEQ ID NO: 4 + SEQ ID


NO: 10 + SEQ ID NO: 11]


(SEQ ID NO: 6)










TAGATCTAAT TGCATCCACT TCGCAGGCAC CTCCTCTGAA TCACCCTCCG




 


CCCTAGACAC GACATGAAGA CTGCATCCAC TGTCGCAGGC ACCTGCCTCT



 


GAATCACCCT CCGCCCTAGA CACGACATGA AGACAGGGGC TGGCGCCAGC



 


CCCTGTCTTC ATGTCGTGTC TAGGGCGGAG GGTGATTCAG AGGCAGGTGC



 


CTGCGACAGT GGATGCAGTC TTCATGTCGT GTCTAGGGCG GAGGGTGATT



 


CAGAGGAGGT GCCTGCGACA GTGGATGCAA TTAGATCTAG CCAGCCCCTG



 


TCTTCATGTC GTGTCTAGGG CGGAGGGTGA TTCAGAGGCA GGTGCCTGCG



 


ACAGTGGATG CAGTCTTCAT GTCGTGTCTA GGGCGGAGGG TGATTCAGAG



 


GCAGGTGCCT GCGACAGTGG ATGCAATTAG ATCTAGGGCT CGAGGTGGGC
(A)


 


GACCCAGACG GGCTTTGACG TCAGCCTGGC CTTTAAGAGG CCGCCTGCCT



 


GGCAAGGGCT GTGGAGACAG AACTCGGGAC CACCAGCTTA AGCTTATCAT
(B)


 


GGAGCAGAAA CTCATCTCTG AAGAAGATCT GGCCCGGGCG TCCATGGAAG



 


ACGCCAAAAA CATAAAGAAA GGCCCGGCGC CATTCTATCC GCTGGAAGAT



 


GGAAAGATGG AACCGCTGGA GAGCAACTGC ATAAGGCTAT GAAGAGATAC



 


GCCCTGGTTC CTGGAACAAT TGCTTTTACA GATGCACATA TCGAGGTGGA



 


CATCACTTAC GCTGAGTACT TCGAAATGTC CGTTCGGTTG GCAGAAGCTA



 


TGAAACGATA TGGGCTGAAT ACAAATCACA GAATCGTCGT ATGCAGTGAA



 


AACTCTCTTC AATTCTTTAT GCCGGTGTTG GGCGCGTTAT TTATCGGAGT



 


TGCAGTTGCG CCCGCGAACG ACATTTATAA TGAACGTGAA TTGCTCAACA



 


GTATCGGCAT TTCGCAGCCT ACCGTGGTGT TCGTTTCCAA AAAGGGGTTG



 


CAAAAAATTT TGAACGTGCA AAAAAAGCTC CCAATCATCC AAAAAATTAT



 


TATCATGGAT TCTAAAACGG ATTACCAGGG ATTTCAGTCG ATGTACACGT



 


TCGTCACATC TCATCTACCT CCCGGTTTTA ATGAATACGA TTTTGTGCCA



 


GAGTCCTTCG ATAGGGACAA GACAATTGCA CTGATCATGA ACTCCTCTGG



 


ATCTACTCGT CTGCCTAAAG GTGTCGCTCT GCCTCATAGA ACTGCCTGCG



 


TGAGATTCTC GCATGCCAGA GATCCTATTT TTGGCAATCA AATCATTCCG



 


GATACTGCGA TTTTAAGTGT TGTTCCATTC CATCACGGTT TTGGAATGTT



 


TACTACACTC GGATATTTGA TATGTGGATT TCGAGTCGTC TTAATGTATA



 


GATTTGAAGA AGAGCTGTTT CTGAGGAGCC TTCAGGATTA CAAGATTCAA



 


AGTGCGCTGC TGGTGCCAAC CCTATTCTCC TTCTTCGCCA AAAGCACTCT



 


GATTGACAAA TACGATTTAT CTAATTTACA CGAAATTGCT TCTGGTGGCG



 


CTCCCCTCTC TAAGGAAGTC GGGGAAGCGG TTGCCAAGAG GTTCCATCTG



 


CCAGGTATCA GGCAAGGATA TGGGCTCACT GAGACTACAT CAGCTATTCT



 


GATTACACCC GAGGGGGATG ATAAACCGGG CGCGGTCGGT AAAGTTGTTC



 


CATTTTTTGA AGCGAAGGTT GTGGATCTGG ATACCGGGAA AACGCTGGGC



 


GTTAATCAAA GAGGCGAACT GTGTGTGAGA GGTCCTATGA TTATGTCCGG



 


TTATGTAAAC AATCCGGAAG CGACCAACGC CTTGATTGAC AAGGATGGAT



 


GGCTACATTC TGGAGACATA GCTTACTGGG ACGAAGACGA ACACTTCTTC



 


ATCGTTGACC GCCTGAAGTC TCTGATTAAG TACAAAGGCT ATCAGGTGGC



 


TCCCGCTGAA TTGGAATCCA TCTTGCTCCA ACACCCCAAC ATCTTCGACG



 


CAGGTGTCGC AGGTCTTCCC GACGATGACG CCGGTGAACT TCCCGCCGCC



 


GTTGTTGTTT TGGAGCACGG AAAGACGATG ACGGAAAAAG AGATCGTGGA



 


TTACGTCGCC AGTCAAGTAA CAACCGCGAA AAAGTTGCGC GGAGGAGTTG



 


TGTTTGTGGA CGAAGTACCG AAAGGTCTTA CCGGAAAACT CGACGCAAGA



 


AAAATCAGAG AGATCCTCAT AAAGGCCAAG AAGGGCGGAA AGATCGCCGT



 


GTAATTCTAG AGTCGGGGCG GCCGGCCGCT TCGAGCAGAC ATGATAAGAT
(C)


 


ACATTGATGA GTTTGGACAA ACCACAACTA GAATGCAGTG AAAAAAATGC



 


TTTATTTGTG AAATTTGTGA TGCTATTGCT TTATTTGTAA CCATTATAAG



 


CTGCAATAAA CAAGTTAACA ACAACAATTG CATTCATTTT ATGTTTCAGG



 


TTCAGGGGGA GGTGTGGGAG GTTTTTTAAA GCAAGTAAAA CCTCTACAAA



 


TGTGGTA



 


 


(Underline (A): SEQ ID NO: 4 = TH gene promoter)



(Underline (B): SEQ ID NO: 10 = c-myc tag-luciferase



fusion gene)



(Underline (C): SEQ ID NO: 11 = SV40 virus late poly



(A) addition signal)







The DNA represented by SEQ ID NO:6 includes, in a functionally linked relationship, the nucleotide sequence represented by SEQ ID NO:5 including the enhancer and the promoter linked together; the c-myc-tagged (N-terminus) luciferase gene represented by SEQ ID NO: 10; and the SV40 virus late poly(A) addition signal represented by SEQ ID NO: 11. The DNA also includes a linking moiety having a linker sequence between SEQ ID NO: 5 and SEQ ID NO: 10 and another linking moiety having another linker sequence between SEQ ID NO: 10 and SEQ ID NO: 11.
[Genetically Modified Animal]
The genetically modified mouse may be obtained by a process that includes introducing the DNA into a mouse and selecting a mouse having the DNA in its genome. In an embodiment of the invention, the DNA to be introduced into a mouse preferably, but not exclusively, has a homology of at least 80% to SEQ ID NO:6. The DNA does not necessarily have a homology of at least 80% to SEQ ID NO: 6, as long as it includes a functionally linked enhancer, promoter, and reporter gene, and as long as the enhancer includes at least one AhR-binding enhancer. The mouse to be transfected with the DNA is preferably an inbred line such as C57BL/6, DBA2, or Balb/c.
Besides mice, the animals to be transfected with the DNA may include rats, hamsters, and rabbits. In that case, the aryl hydrocarbon receptor-binding enhancer located 5′-upstream of a tyrosine hydroxylase gene, any promoter, the reporter gene, and the poly(A) addition signal may be selected depending on the species of the animal to be transfected with the DNA, and DNA formed by functionally linking them may be used.
[Introduction of DNA]
The DNA is most generally introduced into an animal by microinjection method. Specifically, a small amount of the DNA including the functionally linked enhancer, promoter, reporter gene, and transcription termination signal sequence is injected under a microscope into the male pronucleus of a fertilized egg at the 1-cell stage using a micromanipulator.
The surviving fertilized egg is transplanted into a pseudopregnant parent oviduct. The mouse with the transplant is then fed, and an individual carrying the introduced DNA is selected from the offspring mice by Southern blotting, PCR and so on. For example, the genome DNA obtained from the tail of each resulting mouse is analyzed by Southern blotting, and the individual mouse inheriting the introduced DNA is selected. This means that the next generation inherits the introduced DNA and supports the production of genetically modified mice useful as animal models.
Other methods include methods using DNA-containing virus vectors and methods using ES cells. The virus vector method may include removing a pellucid zone from an embryo at the 4- to 8-cell stage, infecting the embryo with a virus vector, then developing the embryo into a blastocyst, transferring the blastocyst into a pseudopregnant mother, and developing the blastocyst so that a genetically modified mouse individual can be obtained.
In the method using ES cells (embryonic stem cells), chimeric mice may be produced from DNA-transfected ES cells, and then genetically modified mouse individuals may be obtained.
Homozygous mice having the introduced gene in both homologous chromosomes may be obtained, and the resulting male and female mice may be mated so that genetically modified mice can be reproduced from generation to generation in such a manner that all offspring can stably carry the gene. The fertilized egg for use in maintaining the genetic traits of the genetically modified mouse may be obtained by mating male and female mice. The fertilized egg may also be obtained by natural mating. However, it is preferred that the estrous cycle of female mice be artificially regulated, and then male mice be mated with the female mice.
In an aspect of the invention, there is provided a genetically modified mouse having a central nervous system in which an indicator trait (reporter) is expressed in such a manner that a general-purpose enzyme-activity-detecting reagent kit and a general-purpose measuring apparatus can be used. The use of the genetically modified mouse allows easy and fast in vivo evaluation of the effect of the test substance on the central nervous system. Specifically, the genetically modified mouse according to the invention has the property of expressing the reporter gene in the central nervous system and is useful as a biological testing material for evaluating the effect of the test substance on the central nervous system, because the toxicity of the test substance can be examined by biochemical quantification using the expression of the reporter gene as an indicator.
[Method for Evaluating the Effect of the Test Substance]
An embodiment of the invention is directed to a method that includes administering a test substance to the genetically modified mouse of the invention and then measuring the expression of the reporter gene in the central nervous system of the genetically modified mouse to evaluate the effect of the test substance on the central nervous system.
When the reporter gene is a firefly luciferase gene, the method may include administering luciferin, a substrate for the luciferase, by intraperitoneal injection to the genetically modified mouse 6 to 72 hours after the administration of the test substance to the genetically modified mouse and measuring luminescence derived from the individual or derived from the enzyme activity of the luciferase in the brain with a luminescence measurement system such as an In-Vivo Imaging System (IVIS), manufactured by Xenogen Corporation.
Alternatively, the method may include harvesting the brain from the genetically modified mouse, extracting proteins from the brain with an appropriate protein extraction reagent, adding luciferin to the extract, and then measuring the degree of luminescence derived from the enzyme activity of the luciferase with a commercially available luminometer or the like. In the measurement, when the test substance-administered genetically modified mice significantly differ in the degree of luminescence from the control group genetically modified mice to which no test substance is administered, the test substance is evaluated to have an effect on the central nervous system. At this time, the amount of change in the degree of luminescence caused by the administration of the test substance may be used as an index for determining the magnitude of the effect of the test substance on the central nervous system.
Another embodiment of the invention is directed to a method that includes isolating primary neuronal cells from the genetically modified mouse, exposing the primary neuronal cells to a test substance, and then measuring the expression of the reporter gene in the cells to evaluate the effect of the test substance on the central nervous system. When the reporter gene is a firefly luciferase gene, the method may include isolating primary neuronal cells from the genetically modified mouse, exposing the primary neuronal cells to a test substance, collecting the cells to extract proteins therefrom 2 to 72 hours after the exposure, adding luciferin, a substrate for the luciferase, to the liquid extract, and measuring luminescence derived from the enzyme activity of the luciferase with a luminometer.
In the evaluation, when the primary neuronal cells from the test substance-administered genetically modified mice significantly differ in the degree of luminescence from the primary neuronal cells from the control group genetically modified mice to which no test substance is administered, the test substance is evaluated to have an effect on the central nervous system. At this time, the amount of change in the degree of luminescence caused by the administration of the test substance may be used as an index for determining the magnitude of the effect of the test substance on the central nervous system.
In this context, the “test substance” may be a substance having dioxin-like activity. The inventors have found and already reported that substances having dioxin-like activity can act on the above enhancer region to enhance the activity of the downstream gene. It has been concluded that substances having dioxin-like activity can form a complex with an aryl hydrocarbon receptor, so that the complex can bind to the enhancer region to enhance the activity of the transcription of the downstream gene.
FIG. 1 shows an example of the mechanism of the gene transcription activation by the aryl hydrocarbon receptor (AhR). It is known that dioxins such as TCDD bind to the intracellular AhR to form a complex which binds to a specific sequence of a gene to activate the expression of the downstream gene.
In FIG. 1, Arnt represents an aryl hydrocarbon receptor nuclear translocator. It is known that the AhR that binds to a chemical substance having dioxin-like activity forms a complex with the Arnt, so that the AhR binds to the enhancer to activate the expression of the downstream gene.
According to the invention, the method for determining the harmfulness of a test substance with the genetically modified animal may include bringing the test substance into contact with the genetically modified animal and detecting the expression of the reporter gene.
For example, the contact may be oral administration of the test substance or parenteral administration such as intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection, intracutaneous injection, transvaginal administration, transintestinal administration, transdermal administration, transnasal administration, transpulmonary administration, or instillation, of the test substance. The exposure to the test substance may also be performed by the same method as the contact method.
After the contact, the presence or absence of the expression of the reporter gene or the magnitude thereof may be detected or measured by a method suitable for the expressed reporter gene. In this case, the whole of the animal may be subjected to observation and/or measurement, or the brain may be removed and subjected to a process such as homogenization, cutting, mincing, and/or protein extraction as desired, followed by observation and/or measurement.
Alternatively, the method for determining the harmfulness of a test substance with the genetically modified animal may be performed by bringing a test substance into contact with part of the genetically modified animal. In this case, the test substance may be brought into contact with part of the genetically modified animal, such as a tissue or an organ. Thereafter, the presence or absence of the expression of the reporter gene or the magnitude thereof may be detected or measured by a method suitable for the expressed reporter gene.
While genetically modified mice have been predominantly described as a typical example of the genetically modified animal, animals other than mice may also be transfected with the DNA by known methods in the same manner so that genetically modified animals can be produced, and any of the features described above may also be applied as in the case of mice. In addition, any of such animals may also be used in the method for determining the harmfulness of a test substance as in the case using the mouse.
EXAMPLES
The invention will be more specifically described by the examples below, which are only for illustration and not intended to limit the scope of the invention.
(1) Preparation of Vector to be Introduced into Mouse
A single-stranded DNA was synthesized in which the −175 by to −237 by region located 5′-upstream of the TH gene and containing the sequence shown in SEQ ID NO: 1 was repeated twice (namely, containing two AhR-binding enhancers (SEQ ID NO: 1) for the TH gene). PCR was performed using the DNA as a template and using specific primers so that the single-stranded DNA was converted to a double strand. The single-stranded DNA (SEQ ID NO: 12) and the nucleotide sequences of the specific primers (SEQ ID NO: 13 and NO: 14) are shown below.







Single-stranded DNA:


(SEQ ID NO: 12)


5′-GCCAGCCCCTGTCTTCATGTCGTGTCTAGGGCGGAGGGTGATTCAGA


 


GGCAGTGCCTGCGACAGTGGATGCAGTCTTCATGTCGTGTCTAGGGCGGA


 


GGGTGATTCAGAGGCAGGTGCCTGCGACAGTGGATGCAATTAGATCT


 


A-3′


 


Primer (forward):


(SEQ ID NO: 13)


5′-GCCAGCCCCTGTCTTC-3′


 


Primer (reverse):


(SEQ ID NO: 14)


5′-TAGATCTAATTGCATC-3′


 


[SEQ ID NO: 12]


TH gene AHR-binding enhancer (single strand, SEQ


ID NO: 2 x2)


GCCAGCCCCT GTCTTCATGT CGTGTCTAGG GCGGAGGGTG


 


ATTCAGAGGC AGTGCCTGCG ACAGTGGATG CAGTCTTCAT


 


GTCGTGTCTA GGGCGGAGGG TGATTCAGAG GCAGGTGCCT


 


GCGACAGTGG ATGCAATTAG ATCTA


 


[SEQ ID NO:13]


Primer: TH gene AHR-binding enhancer forward


GCCAGCCCCT GTCTTC


 


[SEQ ID NO: 14]


Primer: TH gene AHR-binding enhancer reverse


TAGATCTAAT TGCATC






The double-stranded DNA obtained by the PCR was phosphorylated with T4 polynucleotide kinase and then ligated with T4 DNA ligase. A DNA fragment having three linked units was separated by agarose gel electrophoresis and then purified using QIAquick Gel Extraction Kit (QIAGEN). The purified DNA fragment included six repeated AhR-binding enhancers. The DNA was incorporated into the vector PGV-P2 (Toyo B-Net Co., LTD.), a luciferase expression vector. First, the SV40 promoter on the PGV-P2 was cut with the restriction enzymes Hind III and Xho I and replaced with a TH gene promoter (the region from the transcription start point (0 bp) to −100 by (5′-upstream) (SEQ ID NO: 4)) so that a recombinant vector (PGV-THp) was prepared. The c-myc tag sequence shown below was then inserted between the Hind III and Nco I sites of the PGV-THp.








c-myc tag sequence (forward)



(SEQ ID NO: 8)



5′-AGCTTATCATGGAGCAGAAACTCATCTCTGAAGAAGATCTGGCCCGGGCGTC-3′



 


c-myc tag sequence (reverse)


(SEQ ID NO: 9)



5′-GATGGACGCCCGGGCCAGATCTTCTTCAGAGATGAGTTTCTGCTCCATGATA-3′







The c-myc tag sequence was obtained by annealing the complementary single-stranded DNAs. In order to facilitate the incorporation into the vector, a Hind III recognition sequence (5′-end) and a Nco I recognition sequence (3′-end) were added to the ends of the c-myc tag. The resulting double-stranded c-myc tag sequence was incorporated between the Hind III and Nco I sites of the PGV-THp. The vector obtained by this process, namely, the vector PGV-THp-Mluc, in which the c-myc tag sequence was added in-frame to the N-terminus of the luciferase gene on the PGV-THp, was cut with Sma I. The DNA sequence (SEQ ID NO: 5) including the six repeated AhR-binding enhancers and the TH gene promoter was inserted into this site so that the vector pTHEn-MLuc to be introduced into mice was prepared.
The nucleotide sequence from the AhR-binding enhancer to the c-myc tag sequence on the pTHEn-MLuc was checked by sequencing. The vector preparation flow is shown in FIG. 2.
(2) Production of Genetically Modified Mouse Candidates
C57BL6-strain mice were used. Three days before gene introduction, serum gonadotropin (PMSG) was intraperitoneally injected into 4- to 6-week-old female mice, and chorionic gonadotropin (hCG) was then intraperitoneally injected the day before the gene introduction. After the injection, the female mice were mated with male mice, and the next day, the fallopian tube was removed from female mice having a vaginal plug. The ampulla of the fallopian tube was broken in a hyaluronidase-containing culture solution so that the egg mass was removed. The egg mass was allowed to stand for a while so that fertilized eggs were dissociated from cumulus cells. The fertilized eggs were then collected using an egg collecting pipette. The collected fertilized eggs were washed several times with a culture solution and then cultured in a CO2 incubator.
The gene to be introduced into the fertilized eggs was prepared by a process including cutting the DNA represented by SEQ ID NO: 6 (see FIG. 3, TH gene enhancer and promoter::c-myc-tagged luciferase gene::SV40 poly(A) signal) from the pTHEn-MLuc with the restriction enzymes Kpn I and Bam HI, purifying the DNA and dissolving the DNA in a TE buffer (10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA) at a concentration of 1 to 2 ng/μl.
The fertilized eggs were taken out of the CO2 incubator and placed in a medium in a chamber mounted on a microscope. The fertilized egg was held with a holding pipette under low magnification, and then the DNA represented by SEQ ID NO: 6 prepared by the method described above was introduced into the fertilized egg with an injection pipette under a high-power differential interference lens. After the medium was replaced, the fertilized eggs after the injection were cultured in the CO2 incubator for 24 hours. The resulting fertilized eggs were transplanted into pseudopregnant female mice and developed so that genetically modified mouse candidates were produced.
(3) Selection of Genetically Modified Mice by Genotyping
Genetically modified mice were selected from the mouse candidates (prepared in the section (2)) using Southern blotting and PCR to detect part of the introduced DNA (SEQ ID NO: 6, the DNA introduced into the fertilized egg) on the genome. An about 2 cm piece was cut from the tail of each mouse (4 to 6 weeks old) and placed in a centrifuge tube. After the cut piece was treated with protease K at 50° C. for 8 to 10 hours, the same amount of a mixture of phenol, chloroform and isoamyl alcohol (25:24:1) was added to the solution and mixed together. The resulting mixture was centrifuged at 15,000 rpm for 3 minutes to be separated into an aqueous layer and a phenol layer. A new tube was attached to the aqueous layer, and 1⁄4 amount of 10 M ammonium acetate and 2.5 times amount of ethanol were added and mixed well. The mixture was then centrifuged at 15,000 rpm for 3 minutes so that the genome DNA was precipitated from the solution and the supernatant was removed. The precipitate was washed with 70% ethanol and then air-dried. The precipitate was dissolved in the TE buffer again so that a mouse genome DNA solution was obtained.
The process of genotyping by Southern blotting included adding the restriction enzyme Xho I to the genome DNA solution to completely digest the DNA, subjecting the digest to agarose gel electrophoresis, and then transferring the genome DNA in the gel to a nylon membrane Hybond N+ (GE Healthcare Bioscience) by a capillary transfer method. The DNA was fixed on the membrane by ultraviolet irradiation. The membrane was immersed in a hybridization buffer ( 1/20 amount of the Denhardt's solution, 50 μg/ml of salmon sperm DNA), and pre-hybridization was performed at 65° C. for 1 hour. Hybridization was then performed at 65° C. overnight in a hybridization buffer containing a radioisotope (32P)-labeled probe DNA. The non-specifically adsorbed probe was removed by washing the membrane in washing solution A (2×SSC, 0.1% SDS) at 65° C. for 5 minutes twice and then washing the membrane in washing solution B (0.5×SSC, 0.1% SDS) at 65° C. for 15 minutes twice. A radioisotope (32P)-labeled Hind III-Sph I fragment of the pTHEn-MLuc was used as a hybridization probe. The hybridization signals were detected using an X-ray film. As a result, signals derived from the introduced DNA were detected in three genetically modified mouse candidates #176, #177 and #184 (FIG. 4).
The process of genotyping by PCR included determining whether or not part of the luciferase structural gene of the pTHEn-MLuc was amplified using specific primers and using, as a template, the genome DNA extracted from the mouse tail. The nucleotide sequences of the primers used are shown below (SEQ ID NO: 15 and SEQ ID NO: 16).









Primer (forward):







(SEQ ID NO: 15)









5′-GCGAAGGTTGTGGATCTGGATACC-3′


 



Primer (reverse):







(SEQ ID NO: 16)









5′-CCTTTCGGTACTTCGTCCACAAAC-3′


 







[SEQ ID NO: 15]









Primer: luciferase forward



GCGAAGGTTG TGGATCTGGA TACC


 







[SEQ ID NO: 16]









Primer: luciferase reverse



CCTTTCGGTA CTTCGTCCAC AAAC






The amplification was checked by agarose gel electrophoresis. As shown in FIG. 5, signals derived from the introduced DNA were detected in four genetically modified mouse candidates #176, #177, #178, and #184 by PCR genotyping.
The three mice (#176, #177 and #184) in which the introduced DNA signal was detected by both of the two genotyping methods, namely both of the Southern hybridization method and the RCR method, were selected as genetically modified mice and used in the later experiments. Fertilized eggs and sperms from the mouse #176 were cryopreserved and deposited with the International Patent Organism Depositary (Accession No. FERM BP-10993).
(3) Conservation of Genetically Modified Mouse and Line
The three genetically modified mice selected in the section (2) (the 0th generation of the genetically modified mouse, T0 generation) were mated with C57BL/6-strain wild-type mice (back cross) to produce offspring. The offspring of the genetically modified mice were mated with C57BL/6-strain wild-type mice or the offspring of the genetically modified mice so that the genetically modified mice were conserved.
(4) Measurement of Luciferase Luminescence in Genetically Modified Mouse During Exposure to βNF
A dioxin analog, β-naphthoflavone (βNF), was used as a test substance. A solution of βNF in corn oil was intraperitoneally injected at a βNF dose of 40 mg/kg into each of the genetically modified mice #176 (4 weeks old), #177 (6 weeks old) and #184 (6 weeks old) in the T1 generation. The same amount of corn oil as the amount of the solution in the βNF-administered group was intraperitoneally administered to each of the control group mice (the genetically modified mice in the T1 generation). A luciferin solution was intraperitoneally administered at a dose of 150 mg/kg BW to these mice 72 hours after the above administration. After 5 minutes, an In-Vivo Imaging System (IVIS 100-series), manufactured by Xenogen Corporation, was used to detect luciferase luminescence in the mice. As a result, luciferase luminescence was observed in all the genetically modified mice (FIG. 6). In addition, the brain of the genetically modified mouse #176 was cut in the vicinity of the midbrain, and luminescence from the cut section was measured with the IVIS System. As a result, an increase in the degree of luminescence was detected in the vicinity of the midbrain or hippocampus of the βNF-administered mouse (FIG. 7). The results showed that when the change in the expression of the reporter gene was used as an indicator, the genetically modified mice (#176, #177 and #184) made it possible to evaluate the effect of βNF on the central nervous system.
(5) Measurement of Luciferase Luminescence in Primary Neuronal Cells Isolated from Genetically Modified Mouse During Exposure to Dioxin (TCDD)
The genetically modified mouse #176 (hetero, male) was mated with a wild-type mouse, and 12 days after the observation of the plug, the brain was removed from the embryo (E12: an embryonic age of 12 days) under a sterile environment. All the removed embryonic brains were collected, and 0.25% trypsin was added thereto and incubated at 37° C. for 15 minutes. The trypsin was removed. After washed tree times with the Hank's solution, the cells were suspended in a serum-containing DF 1:1 medium (Dulbecco-F12 equal-ratio mixture medium+10% fetal bovine serum). The suspension was inoculated on a culture plate (coated with ornithine-fibronectin) and cultured under a 37° C./5% CO2 atmosphere. After culturing overnight, the medium was replaced with a serum-free medium (Dulbecco MEM/Ham's F12 equal-ratio mixture medium+TIPS (transferrin/insulin/penicillin/streptomycin)) so that the neuronal cells could be predominantly grown (FIG. 8).
After 4 days, TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, Kanto Kagaku) was added at a final concentration of 0, 0.1 or 10 ng/ml to the medium, and after 24 hours, the cells were collected. After the collected cells were washed twice with a phosphate buffer solution, 200 μl of a protein extraction solution (Toyo B-Net Co., Ltd.) was added thereto and stirred at room temperature for 15 minutes. The cells were then broken by freezing (−80° C.) and thawing (room temperature). The broken cell solution was collected in a 1.5 ml tube and then centrifuged so that the supernatant was collected as an extracted protein solution. The resulting extracted protein solution was used in the measurement of luciferase luminescence. In the luciferase luminescence measurement, 100 μl of an assay solution (PicaGene Assay Kit, Toyo B-Net Co., Ltd.) was added to 10 μl of the collected protein solution and stirred, and immediately after the stirring, the degree of luminescence per 2 minutes was measured with a luminometer LB-940 (Berthold). The resulting degree of luminescence was divided by the total protein amount of the protein solution so that it was converted into a relative luminescence intensity (RLU/min/μg protein). The total protein amount of the solution was measured with a protein assay kit (Bio-Rad). As a result, the primary neuronal cells isolated from the genetically modified mouse showed a TCDD concentration-dependent increase in the degree of luminescence (for example, the exposure to 0.1 nM TCDD and 10 nM TCDD produced a 1.3 times increase and a 3.1 times increase from the unexposed case, respectively; FIG. 9). The result showed that when the change in the expression of the reporter gene was used as an indicator, the primary neuronal cells isolated from the genetically modified mouse (#176) made it possible to evaluate the effect of TCDD on the central nervous system.
Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.

## Claims

1. A transgenic animal selected from the group consisting of a mouse, a rat, a hamster, and a rabbit, whose genome comprises a heterologous DNA comprising an aryl hydrocarbon receptor-binding enhancer operably linked to a promoter, a reporter gene, and a poly(A) addition signal; wherein the aryl hydrocarbon receptor-binding enhancer comprises the polynucleotide sequence of SEQ ID NO: 3.

2. The transgenic animal according to claim 1, which is a mouse.

3. The transgenic animal according to claim 2, wherein the promoter consists of SEQ ID NO: 4.

4. The transgenic animal according to claim 2, wherein the polynucleotide sequence of the enhancer and the promoter linked together consists of the polynucleotide sequence of SEQ ID NO: 5.

5. The transgenic animal according to claim 2, wherein the heterologous DNA comprising an aryl hydrocarbon receptor-binding enhancer operably linked to a promoter, a reporter gene, and a poly(A) addition signal consists of SEQ ID NO: 6.

6. The transgenic animal according to claim 5, having Accession No. FERM BP-10993.

7. The transgenic animal according to claim 1, which is a rat.

8. The transgenic animal according to claim 1, wherein the reporter gene is selected from the group consisting of a luciferase gene, a green fluorescent protein gene, a β-galactosidase gene, and a chloramphenicol acetyltransferase gene.

9. The transgenic animal according to claim 8, wherein the reporter gene is a luciferase gene.

10. The transgenic animal according to claim 1, wherein expression of the reporter gene is induced in a central nervous system.

11. A method for determining harmfulness of a test substance, comprising:
bringing the test substance into contact with the transgenic animal according to claim 1 or part thereof; and
detecting expression of a reporter gene.

12. A method for determining harmfulness of a test substance, comprising:
bringing the test substance into contact with the transgenic animal according to claim 9 or part thereof; and
detecting expression of a luciferase gene.

13. A method for detecting an effect of a test substance on a dopamine synthesis, comprising bringing the test substance into contact with the transgenic animal according to claim 1 or part thereof
detecting expression of a luciferase gene.

14. A method for determining an effect of a test substance on a dopamine synthesis, comprising:
bringing the test substance into contact with the transgenic animal according to claim 9 or part thereof; and
detecting test substance-induced expression of a luciferase gene.