JPH09322675A - Animal for transferring thyroid hormone receptor gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nonhuman mammal having DNA integrated with an alien thyroid hormone receptor gene (modifier gene) and useful for elucidating a critical mechanism of a thyroid hormone insensitiveness and as a curing model and for screening of a therapeutic agent of a disease of the thyroid. SOLUTION: This nonhuman mammal of a rodent, etc., such as a mouse or a rat having DNA integrated with an α- or a β-type, etc., of alien thyroid receptor gene (modifier gene) is utilized for elucidating a pathological state mechanism of a hyperthyroidism, a Basedow disease, a thyroiditis, a goiter and a thyroid cancer, etc. Preferably, a gene therapy of a disease of the thyroid containing a thyroid hormone insensitiveness is performed by using a vector containing the alien thyroid hormone receptor gene (modifier gene) and capable of manifesting in a mammal.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a thyroid hormone receptor gene-transferred animal.

[0002]

BACKGROUND OF THE INVENTION Transgenic animals have a gene introduced into the germ line of the animal or an ancestor of this animal at an early (usually single cell) developmental stage. Wagner
er) [1981, Proceedings of National Academy of Science (Proc. Nat. Acad. S
c. USA, 78, 5016] and Stewart et al. [1982, Science, 217].
Vol. 10, page 1046] describes transgenic mice containing the human globin gene. Constantine
tantini) et al. [1981, Nature, Vol. 294,
Page 92] and Lacy et al. [1983, Cell]
34, 343] describe transgenic mice containing the rabbit globin gene. McKnight (Mc
Knight, et al. [1983, Cell Vol. 34, p. 335] describe transgenic mice containing the transferrin gene. Brinstar et al. [1983, Nature, Vol. 306, p. 332], describe transgenic mice containing functionally transposed immunoglobulin genes. Palmiter etc. [l98
2, Nature, Vol. 300, p. 6ll], describes transgenic mice containing a rat growth hormone gene linked to a heavy metal-inducible metallothionein promoter sequence. Palmiter, etc. [1982, Cell]
29, 701] describes transgenic mice containing a thymidine quinase gene linked to a metallothionein promoter sequence. Palmiter, etc. [1983,
Science, Vol. 222, p. 809] describes transgenic mice that contain the human growth hormone gene fused to a metallothionein promoter sequence.

Thyroid hormone receptors are known to form dimers and bind to target genes to control transcriptional activity. An important disease of the thyroid hormone receptor is thyroid hormone refractory disease. It indicates a pathological condition in which the responsiveness to thyroid hormone is reduced due to an abnormality in the target tissue, and there are those in which the peripheral metabolic state is normal or lowered (systemic type) and those in which it is accelerated (pituitary type). Usara et al. [1988, (Mol. Endocrinol.) Volume 2, Vol.
[1217]] is a restriction fragment polymorphism (generally abbreviated as RFLP) in a family with multiple systemic thyroid hormone refractory diseases.
We conducted a linkage analysis by the method and found that the thyroid hormone receptor gene β ₁ was linked to the refractory phenotype. Sakurai et al. [1989, Proceedings of National Academy of Science (Proc. Nat.
Acad. Sc. USA,) 86, 8977] is the isolation of a complementary DNA of the thyroid hormone receptor gene β ₁ from fibroblasts of a patient with systemic thyroid hormone refractory disease, and its nucleotide sequence is examined. To 1318 amino acid, guanine, was mutated to cytosine. After that, the inventor (Nakamura) et al. (1992, Molecular and Seller End Clinology (Mol. Cell Endocr
inol) Vol. 84, p. 159) is based on a survey of patient pedigrees, and the 1612th amino acid from the N-terminus, adenine, is replaced with guanine.
Furthermore, it was discovered that the 438th amino acid, lysine, was mutated to glutamic acid. The intracellular distribution and expression level of thyroid hormone receptor protein in the fibroblasts of this patient are normal, but the thyroid hormone T ₃ binding affinity constant of the mutant thyroid hormone receptor is reduced to 1/20 or less. I found it. Still other researchers have reported numerous abnormalities in the thyroid hormone receptor gene β ₁ , all of which are restricted to its T ₃ -binding region. Inventor (Nakamura) et al. [1993, Journal of Clinical Endocrinology and Metabolism (J. Clin. Endocrinol. Metab.) Vol. 76, No. 12]
[54] also discovered the first thyroid hormone receptor abnormality in a patient with pituitary thyroid hormone refractory disease. Also,
Thyroid hormone receptors have α-type and β-type, but in actual cases, β-type is abnormal in all cases. In addition, thyroid hormone refractory is characterized as a heterozygous disease in which normal and abnormal thyroid hormone receptors are present. In the case, the largest inhibitory effect on the normal receptor was confirmed in the abnormal receptor in which 11 amino acids were deleted from the C-terminal of the abnormal thyroid hormone receptor. In all cases, diffuse goiter of any type is observed, follicular epithelial cell hyperplasia is observed in the thyroid tissue, and the thyroid gland is called thyroid stimulating hormone (generally abbreviated as TSH). It is known to be stimulated by. In addition, inappropriate secretion of thyroid stimulating hormone (syndro
meof inappropriate secretion of TSH, commonly abbreviated as SITSH. If a) is mild, goiter may not be present. However, the mechanism of normal receptor function inhibition by this abnormal receptor and the mechanism of action of thyroid hormone refractory have not been clarified yet.

[0003]

Therefore, if a thyroid hormone receptor gene-transferred animal having a DNA incorporating an exogenous thyroid hormone receptor gene is successfully prepared,
In normal thyroid hormone receptor gene transfer animals,
We believe that it will be possible to elucidate the function of thyroid hormone receptor, elucidate the mechanism of thyroid disease onset, study treatment methods, supply cells with high expression of normal thyroid hormone receptor gene, and elucidate the target gene regulation mechanism of nuclear receptors. In animals with abnormal thyroid hormone receptor gene transfer, elucidation of the pathogenic mechanism of thyroid hormone refractory disease, investigation of treatment methods, supply of cells with high expression of abnormal thyroid hormone receptor gene, target gene regulation mechanism of nuclear receptor It will be possible to clarify In addition, by using a vector containing an exogenous thyroid hormone receptor gene and capable of being expressed in mammals, gene therapy of thyroid diseases including thyroid hormone refractory is also possible.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that the thyroid hormone receptor gene having an exogenous thyroid hormone receptor gene or a DNA in which a mutant gene thereof is incorporated. As a result of successful production of a metastatic animal and further intensive studies, the present invention has been completed. That is, the present invention

(1) A non-human mammal having a DNA incorporating the exogenous thyroid hormone receptor gene or its mutated gene, (2) the exogenous thyroid hormone receptor gene or its mutated gene is of the α type. The non-human mammal described in (1) above, (3) the non-human mammal described in (1) above, wherein the exogenous thyroid hormone receptor gene or its mutant gene is β-type, and the non-human mammal described above is a rodent. A certain animal according to (1) above, (5) an animal according to (4) above, wherein the rodent is a mouse or rat, (6) containing an exogenous thyroid hormone receptor gene or a mutant gene thereof, The present invention provides an expressible vector, and (7) a drug for gene therapy comprising the vector described in (6) above.

[0006] The transgenic animal of the present invention is preferably used for embryos such as unfertilized eggs, fertilized eggs, sperm and embryonic cells containing progenitors thereof, preferably at the stage of embryogenesis in the development of a non-human mammal (more preferably, , At the stage of single cells or fertilized egg cells and generally before the 8-cell stage), calcium phosphate method, electric pulse method, lipofection method, agglutination method, microinjection method, particle gun method, DE
It is produced by transferring the target thyroid hormone receptor gene by the AE-dextran method or the like.
In addition, by the gene transfer method, somatic cells, living organs,
By transferring the target thyroid hormone receptor gene to tissue cells, etc., it can also be used in cell culture, tissue culture, etc. Furthermore, by fusing these cells with the above-mentioned embryo cells by a cell fusion method known per se. It is also possible to create transgenic animals. The thyroid hormone receptor gene (β ₁ type) was identified in 1989 by Sakurai et al. (Proc. Nat. Acad. Sc. USA, Vol. 86, Vol. 89).
For the first time, complementary DNA was isolated according to [Page 77]. The structure and function of the gene are (1) there is a DNA binding region that binds to a target gene rich in cysteine and a hormone binding region on the carboxyl group terminal side in the central part, and (2) the thyroid hormone receptor has α-type and β-type. There are types, each α ₁
And α ₂ and β ₁ and β ₂ subtypes (of which, the α ₂ type thyroid hormone receptor is an inactive thyroid hormone receptor in which a C-terminal specific amino acid portion is present compared to α ₁ type Known as the body [Izumo, etc.,
Nature 334, 539, 198
8 years], the β ₂ -type thyroid hormone receptor is expressed in cells obtained from rat pituitary gland, and the N-terminal is β ₁ due to alternative splicing.
It is known that it is different from the thyroid hormone receptor [Hodin et al., Science No. 2
44, p. 76, 1989]), (3) There is a thyroid hormone response element (thyroid hormone response element) that binds to the thyroid hormone receptor 5'upstream of various target genes, (4) Thyroid Hormone receptors are either receptor proteins or thyroid hormone receptors auxilary protein
Forms a dimer with (generally abbreviated as TRAP) and binds to the thyroid hormone response element,
(5) A retinoid X receptor (generally abbreviated as RXR) exhibits the same function as TRAP described above. (6) A hydrophobic amino acid repeating structure is present on the carboxyl group terminal side of this receptor, which is used for dimer formation. (7) When the thyroid hormone receptors α ₁ or β ₁ and α ₂ are expressed in the same cell at the same time, which is a involved region, α ₂ currently suppresses the function of the normal thyroid hormone receptor. Are known.

“Non-human mammals” that can be the subject of the present invention
Examples include cattle, pigs, sheep, goats, rabbits, dogs,
Examples include cats, guinea pigs, hamsters, mice, and rats. Among them, rodents that have relatively short ontogeny and biological cycle from the viewpoint of creating a pathological animal model system and are easy to reproduce, especially mice (for example, as pure strains, C57BL / 6 strain, DBA2 strain, etc.) B6C3F ₁ system, BDF ₁ system, B6D2F ₁ system
Strain, BALB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD, etc.) is preferred. The “mammals” that can be targeted in the present invention include humans and the like in addition to the above “non-human mammals”. The thyroid hormone receptor mutant gene of the present invention includes a mutation in the original thyroid hormone receptor gene DNA sequence (for example,
Mutations, etc.), specifically, genes in which base addition, deletion, substitution with other bases, etc. have occurred. The thyroid hormone receptor abnormal gene of the present invention means a gene that expresses an abnormal thyroid hormone receptor, and examples thereof include a gene that expresses a thyroid hormone receptor that suppresses the function of a normal thyroid hormone receptor. To be The exogenous thyroid hormone receptor gene in the present invention may be derived from a mammal of the same species or different species as the target animal. When transferring the thyroid hormone receptor gene to a target animal, it is generally advantageous to use the gene as a gene construct linked downstream of a promoter that can be expressed in animal cells. For example, when transferring the human thyroid hormone receptor gene, various mammals having a thyroid hormone receptor gene highly homologous thereto (rabbit,
Target gene constructs (eg, vectors) in which the human thyroid hormone receptor gene is linked downstream of various promoters capable of expressing the thyroid hormone receptor gene derived from dogs, cats, guinea pigs, hamsters, rats, mice, etc. By microinjection into a fertilized egg of an animal, for example, a mouse fertilized egg, a transgenic mammal highly expressing the thyroid hormone receptor gene can be produced. Thyroid hormone receptor expression vectors include Escherichia coli-derived plasmids, Bacillus subtilis-derived plasmids, yeast-derived plasmids, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus. Are used. Of these, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, and the like are preferably used.

[0008] Examples of the promoter for regulating the gene expression include, for example, viruses (eg, simian virus,
Cytomegalovirus, Moloney leukemia virus, JC
As a promoter of a gene derived from a virus, a breast cancer virus, a poliovirus, etc., various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) and birds (chicken, etc.), albumin , Insulin II, uroplakin I
I, elastase, erythropoietin, endothelin,
Muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophin, tartrate resistance Alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally Tie2
Abbreviated), sodium potassium adenosine 3 phosphatase (generally abbreviated Na, K-ATPase),
Neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, M
HC class I antigen (generally abbreviated as H-2L), H-
ras, renin, dopamine β-hydroxylase, thyroid peroxidase (generally abbreviated as TPO), polypeptide chain elongation factor 1α (EF-1α), β actin, α and β myosin heavy chain, myosin light chain 1 and 2 , Myelin basal protein, thyroglobulin, Thy-1, immunoglobulin, H chain variable region (generally abbreviated as VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, preproenkephalin A, vasopressin, etc. There is a promoter,
Cytomegalovirus promoter and human polypeptide chain elongation factor 1 which are preferably highly expressed systemically
The α (EF-1α) promoter, human and chicken β actin promoters, etc. can be used.

The above vector preferably has a sequence (generally called terminator) that terminates the transcription of the messenger RNA of interest in transgenic mammals. For example, it is derived from viruses, various mammals and birds. The sequence of each gene can be used, and preferably the SV40 terminator of simian virus is used. In addition, splicing signals of each gene, enhancer region, a part of the intron of eukaryotic gene, etc. are located 5'upstream of the promoter region, between the promoter region and the translation region or 3'downstream of the translation region in order to further express the target gene. It is also possible to connect to the. The translation region of the normal thyroid hormone receptor is derived from various mammals (rabbit, dog, cat, guinea pig, hamster, rat, mouse, human, etc.)-Derived liver, kidney, thyroid cells, fibroblast-derived DNA and various commercially available genomic DNA. It can be obtained from the library as a whole or a part of genomic DNA or as a starting material complementary DNA prepared from a liver, kidney, thyroid cell, or fibroblast-derived RNA by a known method. Further, as the foreign abnormal thyroid hormone receptor gene, complementary DNA prepared by a known method from RNA derived from fibroblasts of patients with thyroid refractory can be used as a raw material. Further, a translation region obtained by mutating the translation region of the normal thyroid hormone receptor obtained from the above cells or tissues by the point mutagenesis method can be prepared. The translation region can be prepared as a gene construct that can be expressed in a transgenic animal by a conventional genetic engineering method in which it is linked to the downstream of the promoter and, if desired, the upstream of the transcription termination site. The thyroid hormone receptor gene transfer at the fertilized egg cell stage is ensured to be present in all germinal cells and somatic cells of the target mammal. The presence of the thyroid hormone receptor gene in embryonic cells of the transgenic animal after gene transfer means that all progeny of the transgenic animal carry the thyroid hormone receptor gene in all of its embryonic and somatic cells. . The offspring of this type of animal that inherits the gene has the thyroid hormone receptor gene in all of its germ cells and somatic cells.

It has been confirmed that the exogenous normal thyroid hormone receptor gene-transferred non-human mammal of the present invention stably retains the gene by mating, and can be subcultured in a normal breeding environment as the corresponding gene-bearing animal. I can. The transfer of the thyroid hormone receptor gene at the fertilized egg cell stage is ensured to be present in excess in all germinal cells and somatic cells of the target mammal. Excessive presence of the thyroid hormone receptor gene in germ cells of the created animal after gene transfer means that all the offspring of the created animal have excess of the thyroid hormone receptor gene in all of their germ cells and somatic cells. To do. The offspring of this type of animal that inherited the gene have an excess of the thyroid hormone receptor gene in all of its germ cells and somatic cells. By obtaining a homozygous animal having the transgene on both homologous chromosomes and mating the male and female animals, all the progeny can be passaged so that the offspring have the gene in excess. DN incorporating the normal thyroid hormone receptor gene of the present invention
In the non-human mammal having A, the normal thyroid hormone receptor gene is highly expressed, and by promoting the function of the endogenous normal thyroid hormone receptor gene, finally hyperthyroidism, Graves' disease, May develop thyroid diseases such as thyroiditis, goiter, thyroid cancer,
It can be used as a model animal for the pathological condition. For example, by using the mouse of the present invention, it is possible to elucidate the pathological mechanism of thyroid diseases such as hyperthyroidism, Graves' disease, thyroiditis, goiter, and thyroid cancer, and to study treatment methods for this disease. is there. In addition, since an exogenous normal thyroid hormone gene-transferred mammal has a symptom of an increase in the amount of free thyroid hormone, it can be used in a screening test for a therapeutic drug for the above thyroid diseases.

The non-human mammal having the DNA incorporating the exogenous abnormal thyroid hormone receptor gene of the present invention is:
After confirming that the gene is stably retained by mating, the gene-bearing animal can be subcultured in a normal breeding environment. Furthermore, the target gene can be incorporated into the above-mentioned plasmid and used as a protozoa. A gene construct with a promoter can be prepared by a usual genetic engineering technique. The abnormal thyroid hormone receptor gene transfer at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
The presence of the abnormal thyroid hormone receptor gene in the germinal cells of the created animal after gene transfer means that all the progeny of the created animal have the abnormal thyroid hormone receptor gene in all of its germinal cells and somatic cells. The offspring of this type of animal that inherited the gene have an abnormal thyroid hormone receptor gene in all of its germ cells and somatic cells. By obtaining a homozygous animal having the transgene on both homologous chromosomes and mating the male and female animals, all the offspring can be passaged so as to carry the gene. The non-human mammal having the DNA incorporating the abnormal thyroid hormone receptor gene of the present invention has a high expression of the thyroid hormone receptor gene, and by inhibiting the function of the endogenous normal thyroid hormone receptor gene, It may eventually become thyroid hormone refractory and can be used as a model animal for its pathological condition.For example, using the mouse of the present invention, elucidation of the pathological mechanism of thyroid hormone refractory and treatment of this disease can be performed. It is possible to consider. Further, as a specific availability, a mouse that overexpresses an abnormal thyroid hormone receptor gene can serve as a model for elucidating the inhibition of normal receptor function (dominant negative action) by this abnormal receptor in thyroid hormone refractory disease. Conceivable. In that case, the function of each of thyroid hormone receptors α ₁ , α ₂ , β ₁ , and β ₂ will be elucidated, and further as an experimental model to elucidate the mechanism of heterodimer formation with RXR and the gene regulation mechanism of transcription factors. Expected to be used. Furthermore, since transgenic mammals have diffuse goiter, they can be model animals for goiter. Further, since the transgenic mammal has a symptom of an increase in the amount of free thyroid hormone, it can be used for a therapeutic drug screening test for thyroid refractory disease.

Another possibility of using the above-mentioned two types of transgenic mammals is as a cell source for tissue culture, or by directly analyzing DNA or RNA in the tissue of the transgenic mouse of the present invention, or Analysis of the relationship between the complex action of nuclear receptors and transcription factors by analyzing the protein tissues expressed by the gene, culturing cells of the tissue having the gene by standard tissue culture technology, and using these Then, studying the function of cells from tissues that are generally difficult to culture, such as cells derived from thyroid tissue, screening for drugs that enhance the function of cells by using the cells described above, and mutating thyroid hormone receptors Isolation and purification and antibody production thereof are considered.

Using the above transgenic animal, clinical symptoms of thyroid diseases including thyroid refractory disease can be examined. TSH As the method, for example, thyroid hormones T ₃ of tyrosine and T _{4-triiodo-tyrosine} quantification and free thyroid hormone T ₃ tyrosine and T _{4-triiodo-tyrosine} quantification in blood, thyroid stimulating hormone (generally in the blood
Abbreviated), thyroxine-binding globulin (generally abbreviated as TBG) blood thyroglobulin, assay using anti-thyroglobulin or anti-microsomal antibody, assay using TSH receptor antibody, quantification of blood calcitonin, secondary Quantification of thyroid hormone (generally abbreviated as PTH), parathyroid hormone-related peptide (generally PTHR)
(Abbreviated as P) and the like, which enables more detailed determination of disease symptoms and the degree thereof, and as a result, examination of new therapeutic methods becomes possible. In addition, more detailed pathological findings in each organ of thyroid disease model including thyroid refractory disease due to these gene transfer animals were obtained, development of new treatment methods, and further research and treatment of secondary diseases caused by thyroid disease. Can contribute to.

The thyroid gland or each organ is taken out from these gene-transferred animals, and after finely slicing, a gene-transferred cell released by a protease such as trypsin is obtained.
It is possible to systemize the culture or the cultured cells. Furthermore, cAMP reaction, adenylate cyclase activity measurement, and cAMP phosphodiesterase activity measurement can be performed for the purpose of investigating the signal transduction mechanism involved in the differentiation and proliferation of thyroid cells, and their abnormalities can be investigated. Using these cells, in addition to the above-mentioned assay, tyroglobin biosynthesis research by radioimmunoassay (generally abbreviated as RIA), binding assay of thyroid hormone and thyroid hormone receptor, isolation and mass preparation of thyroid hormone receptor are performed. It is possible and is an effective research material for elucidation of thyroid hormone receptor and thyroid hormone action. In addition, by using these test methods and quantitative methods to develop a therapeutic drug for thyroid disease including thyroid refractory disease using these gene-transferred animals, a therapeutic drug for thyroid disease including effective and rapid thyroid refractory disease can be obtained. It becomes possible to provide a screening method. Furthermore, it is possible to study and develop a gene therapy method for thyroid disease using these gene transfer animals or an exogenous thyroid hormone receptor gene expression vector. In the study of nuclear receptors, a model of inactivation of normal thyroid hormone receptor by abnormal thyroid hormone receptor, a model of active transport of T ₃ thyroxine on cell membrane, a model of energy metabolism in mitochondria, a thyroid hormone receptor gene Models for analyzing structural and functional analyzes and evolution of the v-erbA oncogene family, including thyroid hormone and other genes by the thyroid hormone receptor (eg,
It is possible to use the osteocalcin gene) regulation as a model for elucidating (for example, transcriptional regulation by T ₃ response element) or a model for elucidating the action of a transcriptional activity repressor and a promoter involved in nuclear receptors.

In the present specification and drawings, when an abbreviation is used for a base or the like, IUPAC-IUB Commi
sion on Biochemical Nomen
It is based on the abbreviations based on the “culture” or the abbreviations commonly used in this field, and examples thereof are given below. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid RNA: ribonucleic acid A: adenine T: thymine G: guanine C: cytosine

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the base sequence of the 32-mer primer 1 used in the polymerase chain reaction reaction described in Example 1. [SEQ ID NO: 2] This shows the base sequence of the 35-mer primer 2 used in the polymerase chain reaction reaction described in Example 1. [SEQ ID NO: 3] This shows the base sequence of 21-mer primer 3 used in the polymerase chain reaction reaction described in Example 2. [SEQ ID NO: 4] This shows the base sequence of 21-mer primer 4 used in the polymerase chain reaction reaction described in Example 2. [SEQ ID NO: 5] This shows the base sequence of 21-mer primer 5 used in the polymerase chain reaction described in Example 4. [SEQ ID NO: 6] This shows the base sequence of 21-mer primer 6 used in the polymerase chain reaction reaction described in Example 4.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to these.

[0018]

【Example】

Example 1 1) Construction of plasmid pCMXαF397X containing human mutant thyroid hormone receptor α ₁ gene Plasmid pCMX-hTRα ₁ containing human thyroid hormone receptor α ₁ gene [Nakai et al., Molecular and Cellular Endocrinology (Mol) .Cell.En
docrinol.), Vol. 72, p. 143, 1990] 1
After digesting μg with the restriction enzyme Bam HI, it was purified by a conventional method, and it was digested with the restriction enzyme cleavage site BspMI 32
Primer 1 (5'-AGGAGGCGTACC
TGCTGGCGTCTCGAGCACTAC-3 ′: SEQ ID NO: 1) and 3 containing restriction enzyme cleavage site Xho I
5-mer primer 2 (5'-TCAAAGACCTC)
GAGGAAGAGTGGGGGGTCAGAGTTC-
3 ': 94 ° C for 30 seconds, 55 ° C 1 using SEQ ID NO: 2)
Polymerase chain reaction reaction (generally abbreviated as PCR) in which a cycle of 1 minute and 72 ° C. for 1 minute is repeated 50 times
Then, a reaction product having a size of 193 bp was obtained. Next, the obtained DNA was cleaved with restriction enzymes BspMI and Xho I to obtain a DNA fragment. In addition, the plasmid pCMX
-HTRα ₁ is cleaved with restriction enzymes BspMI and Xho I, and a target DNA pCMX-h containing a human thyroid hormone receptor gene expression unit is prepared in the same manner as above.
A 1.6 kbp fragment was obtained by extracting the TRα ₁ BspMI-Xho I fragment. This fragment was ligated to the PCR product using a Takara ligation kit (manufactured by Takara Shuzo), and this reaction solution was used to prepare E. coli JM1.
09 (Nippon Gene) was transformed to obtain an ampicillin resistant strain. This transformant (Escherich
ia coli JM109 / pCMXαF397X)
Recover the plasmid DNA from the
The plasmid pCMXαF397X (5.7 kbp) was obtained. The base sequence of the PCR product was confirmed by the dideoxy method known per se. The above construction diagram is shown in (FIG. 1). This gene construct was examined by multiple restriction enzyme cleavage and contained no detectable transposition.

2) Construction of plasmid pEF-BOSαF397X having a human mutant thyroid hormone receptor α ₁ gene downstream of the control region of human chromosomal gene Human polypep as a control region of human chromosomal gene
side chainlongation fact
The promoters of or 1α are Mizushima and Nagata (Mizu).
Shima & Nagata [Nucleic Acids Research
arch) Vol. 18, p. 5322, 1990], which was used as a plasmid pEF-BOS. First, pEF-BOS was cleaved with a restriction enzyme Xba I, and the obtained 5.8 kbp Xba I fragment containing the control region was treated with a DNA polymerase Klenow fragment (Takara Shuzo) to make a blunt end. This fragment was dephosphorylated at the 5'end with calf intestinal alkaline phosphatase (Takara Shuzo). Next, pCMXαF397X was digested with restriction enzyme H
The human mutant thyroid hormone receptor α ₁ cDNA region was isolated by cutting with ind III and Bam HI, and blunt-ended by the same method as described above. pEF-BOS-Xba
I blunt fragment (5.8 kbp) and pCMXαF397X
-Hind III-Bam HI blunt fragment (1.7 kb
p) was treated with a Takara ligation kit (Takara Shuzo Co., Ltd.) at 16 ° C. for 60 minutes for ligation, and E. coli JM109 (Nippon Gene Co.) was transformed with this reaction solution to obtain an ampicillin-resistant strain. This transformant (Es
cherichia coli JM109 / pEF-B
The plasmid DNA was recovered from OSαF397X) and digested with restriction enzymes to obtain pCMXαF397X-Hind.
The III-Bam HI blunt fragment is pEF-BOS-Xb.
It was confirmed that the DNA was ligated in the aI blunt fragment in the forward direction, and the plasmid pEF-BOSαF397X (7.0 k
bp) was obtained. The construction diagram of this plasmid is shown in (FIG. 2). This gene construct was examined by multiple restriction enzyme cleavage and contained no detectable transposition.

Example 2 1) Generation of Transgenic Mouse Containing Human Mutant Thyroid Hormone Receptor α ₁ Gene Fusion Downstream of the Control Region of Human Chromosomal Gene The above plasmid pEF-BOSαF397X was Hind.
Cleavage with III and ScaI, 10 μg-1
The concentration was adjusted to 00 μg / ml, and 1 to 2 μl thereof was injected into the male pronucleus of single cell stage mouse eggs fertilized while observing under a microscope. Then, inject the injected egg with Wagner (Wagner).
ner) et al. [1981, Proceedings of National Academy of Science (Proc.
Natl. Acad. Sci. U. S. A. ), 78, 5016], and transplanted into the oviducts of pseudopregnant female mice for implantation. These eggs were obtained by mating C57BL / 6JxDBA2F1 mice. Mouse is a commercial product (CLEA Japan)
After the baby was born, the fostered female mouse was allowed to grow until the baby was weaned.

2) Analysis of Human Mutant Thyroid Hormone Receptor α ₁ Gene Transfer Mouse The DNA was collected from the tail of a 3-week-old offspring and tested by the polymerase chain reaction method.
That is, the 21-mer primer 3 (5'-AGGCTGTGCT in the human mutant thyroid hormone receptor cDNA is used.
GCTAATGTCAA-3 ': SEQ ID NO: 3), and 21-mer primer 4 (5'-AAGAGTGGGGGTGCAG in the human mutant thyroid hormone receptor cDNA).
The polymerase chain reaction was performed using AGTTCG-3 ': SEQ ID NO: 4). DNA for analysis is
DNA was extracted from an approximately 1 cm fragment of the tail by the method described by Hogan et al. [1986, Manipuling Mouse Embryo (Cold Spring Harbor)]. The resulting nucleic acid pellet was washed once in 70% ethanol, dried and resuspended in 200 μl of 10 mM Tris (pH 8.0), 1 mM EDTA. 1 μl of the tail DNA preparation was diluted 50-fold with sterile water, and the primers 1 and 2 were used at 94 ° C.
A PCR reaction in which a cycle of 30 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute was repeated 50 times, and the reaction product was subjected to 4% agarose X.
Electrophoresis through a gel (manufactured by Nippon Gene Co., Ltd.)
Mice showing a DNA band of 43 bp in size were selected. 4 out of 117 offspring that underwent PCR reaction
A DNA band with a size of 243 bp was observed in one animal (one of which died after delivery). Further, using a tail DNA preparation, a fragment (300 bp) obtained by cleaving human mutant thyroid hormone receptor α ₁ complementary DNA with Pst I and Bam HI was analyzed by Southern hybridization with a probe labeled with ³² P by the random prime method. did. Then, 10 μg of the DNA of each of these 4 mice was completely digested with restriction enzyme Eco RI, electrophoresed through a 1.0% agarose gel, and further subjected to Southern (1975, Journal-Moleculer Biology). , Vol. 98, No. 503
Transferred to a nylon filter as described by P.). The filter was hybridized to the probe overnight, washed twice with 2xSSC, 0.1% SDS at room temperature and once with 0.1xSSC, 0.1% SDS at 65 ° C. The result of this Southern hybridization method is that EF-1α injected with all three generated mice was injected.
It was shown to retain _one fusion (Fig. 3). Each number in FIG. 3 indicates an individual number of the human mutant thyroid hormone receptor α ₁ gene transfer mouse. From the above results, it was confirmed that the gene-transferred mice were three individuals with individual numbers T8243-1 (dead individuals), T1054-12, and T1056-9. Subsequently, the weight of the human mutant thyroid hormone receptor α ₁ gene-transferred mouse was measured every 1 to 5 weeks. As a result of weight measurement up to 27 weeks after birth, human mutant thyroid hormone receptor α ₁ gene transfer mouse individual number T
1056-9 (male) showed almost no weight gain after 12 weeks. However, 5 non-transgenic mice on the same litter showed an increase in body weight at the same time, and individual number T1056-9
Was clearly different from the change in body weight.

[Table 1] Table 1 above shows the production and analysis results of the human mutant thyroid hormone receptor α ₁ gene transfer mouse obtained in Example 2. Example 3 1) Plasmid pEF having a human mutant thyroid hormone receptor β ₁ gene downstream of the control region of human chromosomal gene
-Construction of BOSβF451X As a control region of human chromosomal gene, human polypep
side chainlongation facto
The promoter of r 1α is Mizushima and Nagata (Mizus
Hima & Nagata [Nucleic Acids Research
rch) Vol. 18, p. 5322, 1990], which was used as a plasmid pEF-BOS. First, pEF-BOS was cleaved with a restriction enzyme Xba I, and the obtained 5.8 kbp Xba I fragment containing the control region was treated with a DNA polymerase Klenow fragment (Takara Shuzo) to make a blunt end. This fragment was dephosphorylated at the 5'end with calf intestinal alkaline phosphatase (Takara Shuzo). Next, human mutant thyroid hormone receptor β ₁
The plasmid pCMXFβ451X containing the gene was cleaved with the restriction enzymes Hind III and Bam HI to isolate the human mutant thyroid hormone receptor β ₁ cDNA region,
A blunt end was prepared in the same manner as above. pEF-BOS-
Xba I blunt fragment (5.8 kbp) and pCMXβF4
51X-Hind III-Bam HI blunt fragment (1.
7 kbp) was treated with Takara Ligation Kit (Takara Shuzo Co., Ltd.) at 16 ° C. for 60 minutes to allow ligation. Escherichia coli JM109 (Nippon Gene Co., Ltd.) was transformed with this reaction solution to obtain an ampicillin-resistant strain. This transformant (Escherichia coli JM109 / p
The plasmid DNA was recovered from EF-BOSβF451X) and digested with a plurality of restriction enzymes, and the DNA fragment produced was in agreement with the expected size.
It was confirmed that the βF451X-Hind III-Bam HI blunt fragment was ligated in the forward direction in the pEF-BOS-Xba I blunt fragment, and the plasmid pEF-BOS was confirmed.
βF451X (6.6 kbp) was obtained. The construction diagram of this plasmid is shown in (FIG. 4). This gene construct was examined by multiple restriction digests and found that it contained no detectable transposition.

Example 4 1) Generation of Transgenic Mouse Containing Human Mutant Thyroid Hormone Receptor β ₁ Gene Fusion Downstream of the Control Region of Human Chromosomal Gene The above plasmid pEF-BOSβF451X was Hind.
Cleavage with III and ScaI, 10 μg-1
The concentration was adjusted to 00 μg / ml, and 1 to 2 μl thereof was injected into the male pronucleus of single cell stage mouse eggs fertilized while observing under a microscope. Then, inject the injected egg with Wagner (Wagner).
ner) et al. [l98l, Proceedings of National Academy of Science (Proc. Nat
Acad. Sci. USA), Vol. 78, No. 5
Page 016] and transplanted into oviducts of pseudopregnant female mice and implanted. These eggs are C57B
Obtained by mating L / 6JxDBA2F1 mice. As the mouse, a commercially available product (CLEA Japan, Inc.) was used, and after delivery, the reared female mouse was raised until weaning. 2) Analysis of human mutant thyroid hormone receptor β ₁ gene transfer mouse Each offspring was tested by the polymerase chain reaction method using DNA collected from the tail at 3 weeks of age.
That is, human mutant thyroid hormone receptor β ₁ cDNA
21-mer primer 5 (5'-CTGTCTTC in
TTTCAACCTGGAT-3 ′: SEQ ID NO: 5), and 21-mer primer 6 (5′-TTAGAGTTCTTGTG in human mutant thyroid hormone receptor cDNA.
The polymerase chain reaction method was performed using GGGCATTC-3 ': SEQ ID NO: 6). DN for analysis
A is Hogan et al. [1986, Manipille Rating.
DNA was extracted from an approximately 1 cm fragment of the tail by the method described in Mouse Embryo (Cold Spring Harbor)]. The resulting nucleic acid pellet was washed once in 70% ethanol, dried and 200 μl of 1
Resuspended in 0 mM Tris (pH 8.0), 1 mM EDTA. 1 μl of tail DNA preparation was diluted 50-fold with sterile water, using Primer 5 and Primer 6,
5 cycles of 94 ° C for 30 seconds, 55 ° C for 1 minute, 72 ° C for 1 minute
A PCR reaction was repeated 0 times, and the reaction product was electrophoresed through a 4% agarose X (Nippon Gene) gel to select a mouse having a DNA band of 275 bp. A total of 53 pups subjected to PCR reaction showed a DNA band of 275 bp in 9 pups. Furthermore, by using the DNA collected from the tail, a fragment (270 bp) obtained by cleaving the human mutant thyroid hormone receptor β ₁ complementary DNA with Sma I and Bam HI was subjected to a ³² P-labeled probe by the random prime method. analyzed. 10 μg of these 6 DNA preparations were then digested to completion with the restriction enzyme EcoRI, electrophoresed through a 1.0% agarose gel, and Southern (1975,
Journal Morequiller Biology, Volume 98,
(Page 503) and transferred to a nylon filter. The filter was hybridized to the probe overnight and 2x at room temperature in 2xSSC, 0.1% SDS.
Washed twice and 6 in 0.1xSSC, 0.1% SDS
It was washed once at 5 ° C. The results of this Southern hybridization method indicated that 6 out of 9 tested mice retained the injected EF-1β ₁ fusion (FIG. 5). Each number in FIG. 5 indicates the individual number of the human mutant thyroid hormone receptor β ₁ gene transfer mouse.
From the above results, the gene transfer mouse was identified as individual number T9082.
-2, T10121-3, T10122-1, T101
It was confirmed to be 6 individuals of 24-1, T10124-7, and T10126-5. Subsequently, the weight of the human mutant thyroid hormone receptor β ₁ gene-transferred mouse was measured for 1 week-
I went every 5 weeks. As a result of weight measurement up to 27 weeks after birth, almost no increase in body weight was observed after 13 weeks in human mutant thyroid hormone receptor β ₁ gene transfer mouse individual number T10122-1. However, litter-transferred non-transgenic mouse 4
The animals showed weight gain at the same time, and the individual number was T10122.
-1 was clearly different from the weight change.

[Table 2] Table 2 above shows the production and analysis results of the human mutant thyroid hormone receptor β ₁ gene-transferred mouse obtained in Example 4.

[0024]

INDUSTRIAL APPLICABILITY The non-human mammal to which the exogenous thyroid hormone receptor gene of the present invention has been transferred can develop thyroid diseases such as hyperthyroidism, Graves' disease, thyroiditis, goiter, and thyroid cancer. , And can be used as a model animal for the pathological condition. For example, using the mouse of the present invention, it is possible to elucidate the pathological mechanism of these and study the treatment method for this disease. The non-human mammal to which the abnormal thyroid hormone receptor gene of the present invention has been transferred is used as a pathological model of thyroid hormone refractory disease, the elucidation of the onset mechanism thereof, the examination of the treatment method, the above It can be used for the purpose of elucidating the target gene regulation mechanism of supply and nuclear receptors. It is also considered that a vector containing the thyroid hormone receptor gene and capable of being expressed in vivo is used for gene therapy of the thyroid disease.

[0025]

[Sequence list]

Sequence number (SEQ ID NO): 1 Sequence length (SEQUENCE LENGTH): 32 Sequence type (SEQUENCE TYPE): Nucleic acid (nucleic acid) Number of strands (STRADEDNESS): Single-strand (single) Topology (TOPOLOGY): Type of linear sequence (MOLECULAR TYPE): other nucleic acid (other nucl)
eic acid) Antisense (ANTI-SENCE): No Sequence: AGGAGGCGTA CCTGCTGGCG TTCGAGCACT AC 32

Sequence number (SEQ ID NO): 2 Sequence length (SEQUENCE LENGTH): 35 Sequence type (SEQUENCE TYPE): Nucleic acid Number of strands (STRADEDNESS): Single strand topology ( TOPOLOGY): Linear sequence type (MOLECULAR TYPE): Other nucleic acid (other nucl)
eic acid) Antisense (ANTI-SENCE): Yes Sequence: TCAAAGACCT CGAGGAAGAG TGGGGGTCAG AGTTC 35

Sequence number (SEQ ID NO): 3 Sequence length (SEQUENCE LENGTH): 21 Sequence type (SEQUENCE TYPE): Nucleic acid (Nucleic acid) Number of strands (STRADEDNESS): Single-strand (single) Topology ( TOPOLOGY): Linear sequence type (MOLECULAR TYPE): Other nucleic acid (other nucl)
eic acid) Antisense (ANTI-SENCE): No Sequence: AGGCTGTGCT GCTAATGTCA A 21

Sequence ID (SEQ ID NO): 4 Sequence length (SEQUENCE LENGTH): 21 Sequence type (SEQUENCE TYPE): Nucleic acid Number of strands (STRADEDNESS): Single strand topology ( TOPOLOGY): Linear sequence type (MOLECULAR TYPE): Other nucleic acid (other nucl)
eic acid) Antisense (ANTI-SENCE): Yes Sequence: AAGAGTGGGG GTCAGAGTTC G 21

Sequence number (SEQ ID NO): 5 Sequence length (SEQUENCE LENGTH): 21 Sequence type (SEQUENCE TYPE): Nucleic acid (Nucleic acid) Number of strands (STRADEDNESS): Single-strand (single) Topology ( TOPOLOGY): Linear sequence type (MOLECULAR TYPE): Other nucleic acid (other nucl)
eic acid) Antisense (ANTI-SENCE): Yes Sequence: CTGTCTTCTT TCAACCTGGA T 21

Sequence number (SEQ ID NO): 6 Sequence length (SEQUENCE LENGTH): 21 Sequence type (SEQUENCE TYPE): Nucleic acid (Nucleic acid) Number of strands (STRADEDNESS): Single-strand (single) Topology ( TOPOLOGY): Linear sequence type (MOLECULAR TYPE): Other nucleic acid (other nucl)
eic acid) Antisense (ANTI-SENCE): Yes Sequence: TTAGAGTTCT GTGGGGCATT C 21

[0031]

[Brief description of drawings]

FIG. 1 shows the plasmid pCMXαF3 obtained in Example 1.
Construction drawing of 97X.

FIG. 2 is a plasmid pEF-BOSαF having the human mutant thyroid hormone receptor α ₁ gene obtained in Example 1.
Construction diagram of 397X.

FIG. 3 shows the results of Southern hybridization of the human mutant thyroid hormone receptor α ₁ gene-transferred mouse obtained in Example 2.

FIG. 4 shows a plasmid pEF-BOSβF having the human mutant thyroid hormone receptor β ₁ gene obtained in Example 3.
Construction diagram of 451X.

FIG. 5 shows the results of Southern hybridization of the human mutant thyroid hormone receptor β ₁ gene-transferred mouse obtained in Example 4.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 15/09 ZNA A61K 37/02 ABJ // C07K 14/72 9282-4B C12N 15/00 ZNAA

Claims (7)

[Claims] 1. A non-human mammal having a DNA incorporating an exogenous thyroid hormone receptor gene or a mutant gene thereof. 2. The non-human mammal according to claim 1, wherein the exogenous thyroid hormone receptor gene or its mutant gene is α-type. 3. The non-human mammal according to claim 1, wherein the exogenous thyroid hormone receptor gene or its mutant gene is β-type. 4. The animal according to claim 1, wherein the non-human mammal is a rodent. 5. The animal according to claim 4, wherein the rodent is a mouse or rat. 6. A vector containing an exogenous thyroid hormone receptor gene or a mutant gene thereof, which can be expressed in mammals. 7. A drug for gene therapy comprising the vector according to claim 6. [0001]