JP5219185B2 - Multiple cystic kidney model small fish and their use - Google Patents

Description

 The present invention relates to a model fish of multiple cystic kidney disease and use thereof, and more specifically, an adult model of multiple cystic kidney disease and prevention, treatment or symptoms of the onset and progression of multiple cystic kidney disease of the model fish. Regarding utilization for improvement.

 Polycystic Kidney Disease (PKD) in humans includes autosomal recessive polycystic kidney disease (ARPKD) and autosomal dominant polycystic kidney disease (ARPKD). ARPKD), among which ADPKD is one of the common genetic diseases, and it is estimated that about 100,000 to 200,000 people are affected in Japan. ADPKD is a systemic disease. In addition to cysts in the kidney, cysts may occur in the liver, pancreas, spleen, etc., and there is a high frequency of intracranial hemorrhage, and more than half of them have hypertension. To do. Renal cyst formation, the most prominent feature in ADPKD, causes enlarged kidneys and reduced ability to concentrate the kidneys. These symptoms in ADPKD patients progress with age, but due to the lack of effective treatment, they may eventually become renal failure and require renal dialysis and kidney transplantation.

 As the genetic pathogenesis of human ADPKD, loss-of-function, especially the two-hit theory, has been proposed. After inheriting the mutant and wild-type PKD genes from the parents, the somatic mutation occurs in the wild-type PKD gene for some reason during the growth process, so the function of the PKD gene is lost and a cyst is formed and developed. (Non-Patent Document 1).

 PKD1 gene and PKD2 gene have been identified as causative genes of human ADPKD. PKD1 gene located at 16p13.3 on human chromosome 16 and PKD2 gene located at 4q21-23 on human chromosome 4, which are protein polycystin 1 (PC1) and polycystin 2 (PC2), respectively. ) (Non-Patent Document 1). In addition, a causal gene has been cloned from a mouse polycystic kidney model (Non-patent Document 2).

 PKD1 is located on human chromosome 16p13.3, the size of mRNA is about 14 kbp, and it is a huge gene consisting of 46 exons. Its gene product, polycystin-1, is composed of 4320 amino acids, its N-terminus is extracellular and C-terminus is intracellular, and it is a transmembrane protein with 11 transmembrane domains. . It is thought to serve as a sensor that transmits signals from outside the cell into the cell. Pkd1 knockout mice have poor phenotype in heterozygotes, and cause polycystic kidney disease and embryonic lethality in homozygotes (Non-patent Document 3).

PKD2 is located on human chromosome 4 4q21-23, has a 5.4 kbp mRNA, and consists of 15 exons. The gene product polycystin-2 (polycystin-2) is a transmembrane protein consisting of 968 amino acids, present in the cell at both the N-terminal and C-terminal, and having six transmembrane domains between them. It is considered a Ca ²⁺ ion channel (Non-patent Document 1). Pkd2 knockout mice, like Pkd1 knockout mice, lack a specific phenotype in heterozygotes, and form polycystic kidneys in homozygotes, resulting in fetal lethality (Non-patent Document 4). As a difference from Pkd1 knockout mice, visceral inversion occurs in homozygotes of Pkd2 knockout mice (Non-patent Document 5).

Both polycystin 1 and polycystin 2 are localized in the primary cilium existing on the luminal side of tubular epithelial cells, and interact with each other at the C-terminal in the cell. Polycystin 1 captures the flow rate of urine in the tubule as an inclination and transmits it to the conjugated polycystin 2, and the polycystin 2 functions as a Ca ²⁺ channel. Ca ²⁺ influx from outside the cell causes a large amount of Ca ²⁺ release from polycystin 2 in the endoplasmic reticulum. It is speculated that this change in intracellular Ca ²⁺ concentration subsequently controls the expression of various genes involved in differentiation, proliferation, apoptosis, etc., and controls the differentiation and proliferation of tubular epithelium. With either mutation of polycystin 1 or polycystin 2, the ciliary function as a sensor is broken, and an appropriate tubule diameter cannot be maintained, resulting in a cyst (Non-patent Documents 6 and 7).

 The resulting cysts increase with several growth mechanisms, one of which has been reported to involve cAMP. cAMP suppresses the proliferation of normal tubular cells, but cAMP promotes the proliferation of tubular cells obtained from ADPKD patients. It is considered that the action of cAMP has changed from cell growth suppression to cell growth promotion due to the decreased expression of polycystin 1 (Non-patent Document 8). Hormones such as vasopressin, prostaglandin E2, secretin, VIP (vasoactive intestinal polypeptide) increase cAMP and may increase cysts. When OPC31260, a vasopressin V2 receptor inhibitor that suppresses cAMP, was administered to spontaneous cystic kidney rats (with human autosomal recessive polycystic kidney gene abnormality) and Pkd2 genetically engineered mice, renal cAMP content decreased, There are reports that cyst formation has also been significantly reduced (Non-Patent Documents 9 and 10). From these facts, cAMP is likely to be involved in cyst formation, and clinical trials of vasopressin V2 receptor inhibitors for its suppression are beginning in the world.

 On the other hand, although a plurality of knockout mice have been created as a model animal for polycystic kidney disease (Non-patent Document 11), much effort and cost are required for maintenance and breeding of transgenic mice. As already explained, normal knockout mice are embryonic lethal and are not suitable for drug administration studies. In addition, Pkd1 conditional knockout mice that have avoided fetal lethality have been reported (Non-patent Document 12). Furthermore, in the Pkd1 knockout chimeric mouse produced by Nishio et al., Multiple cystic kidneys similar to human ADPKD were observed, and it has been reported that it is useful as a pathological analysis model (Non-patent Document 13).

In recent years, attempts to conduct drug screening using small fish have been successful. Medaka (Oryzias latipes) is a model animal from Japan. There are 48 chromosomes, the genome size is as small as 800Mbp, a quarter of the human genome. Breeding is relatively easy, the generation change time is 2 to 3 months, and it has the advantages suitable for genetic research such as egg laying every day. In zebrafish, many kidney cyst-forming mutants have been discovered (Non-patent Documents 14 and 15), and in medaka, a mutant medaka that develops multiple cystic kidneys has been reported (Non-patent Document 16). ). Furthermore, gene knockdown by morpholino antisense oligonucleotides has been reported for medaka (Non-patent Documents 17 and 18).
N Engl J Med 2004; 350: 151-164 J Formos Med Assoc. 2003 Jun; 102 (6): 367-74. J. Hum Mol Gene 2001; 10: 2385-2396 Nat Genet 2000; 24: 75-78. Curr Biol 2002; 12: 938-943. Bioessays 2004; 26: 844-856. Kidney Int 2000; 57: 1460-1471. Nat Med 2004; 10: 363-364. Nat Med 2003; 9: 1323-1326. Am J Physiol Renal Physiol 2003; 285: F1034-F1049. J Am Soc Nephrol 2004; 15: 3035-3043. J Clin Invest 2005; 115: 910-918. Development 1998; 125: 4655-4667. Genes & Dev 2001; 15: 3217-3229. Kidney Int 2005; 68: 23-34 J Am Soc Nephrol. 2006; 17 (10): 2706-18 Development. 2007 Apr; 134 (8): 1605-15

 In spontaneously occurring cystic kidney rats and Pkd2 gene-engineered mice reported in the reports on cAMP suppression disclosed in Patent Documents 9 and 10, the cysts are mainly distal tubules / collecting tubes in which vasopressin V2 receptor is highly expressed. Is formed. Therefore, the effectiveness of human ADPKD, which is thought to be 70% of cysts derived from proximal tubules, is unclear, and further investigation of effective drugs is required. Model animals are needed.

 However, at present, it cannot be said that a model animal suitable for drug screening that suppresses the onset and progression of human ADPKD has been provided. Moreover, it cannot be said that a model animal suitable for simple and rapid screening is provided.

 That is, for examination of drugs, a model animal for multiple cystic kidney disease is required, but as disclosed in Non-Patent Documents 3 and 4, heterozygous knockout mice of PKD1 gene or PKD2 gene are specific to cystic kidneys. Insufficient expression was lethal in homozygous knockout mice. The Pkd1 conditional knockout mouse disclosed in Non-Patent Document 12 avoiding embryonic lethality has a small number of renal cysts, and the Pkd1 knockout chimeric mouse disclosed in Non-Patent Document 14 is expressed by its chimera rate. There was variation in the mold. Thus, none of the model mice was suitable for drug administration studies.

 In addition, cyst nephrogenic mutants have been discovered in zebrafish and medaka fish, but these causative genes are not homologous to PKD1 and PKD2 genes, but the expression of mRNA encoding polycystin 1 and 2 is abnormal. I know it ’s not allowed. Furthermore, although small fishes such as medaka have the advantages described above, knockout is not possible at this time. Furthermore, it is difficult to systematize the gene knockdown method, and the knockdown medaka disclosed in Non-Patent Document 17 is lethal so as to form a cyst but not to an adult fish.

 Accordingly, an object of the present invention is to provide a model fish suitable for studying a drug effective for human ADPKD and the use thereof. The present invention also provides a genetically modified fish that forms a pathological condition of polycystic kidneys (typically an increase in cystic kidneys), has no mutation in embryonic lethality, and has a mutation in the gene responsible for human ADPKD, and uses thereof This is another purpose. Furthermore, another object of the present invention is to provide a fish that can be systematized in such fish and its use.

 The present inventors have focused on medaka as an animal model that enables drug administration studies to be carried out more easily and at low cost. Therefore, the present inventors cloned medaka PKD1 and PKD2, and further examined possible dominant negative mutants for these genes. According to mouse precedents, mutations in the medaka PKD1 gene and / or PKD2 gene were considered to be lethal in medaka, but surprisingly, the dominant negative transgenic medaka of these genes has its phenotype However, the cysts were not lethal but developed post-adult polycystic kidney disease, and the number of cysts increased with the age of the fish, indicating the onset and progress of human ADPKD. The present inventors have completed the following invention based on these findings. That is, according to the present invention, the following means are provided.

 According to the present invention, a polycystic kidney model small fish, which is a genetically modified fish obtained by introducing a mutant gene of the PKD2 gene so as to express a dominant negative effect, is provided. Preferably, the model small fish is a medaka.

 The mutant gene can be a mutant gene of the PKD2 gene derived from the same species of fish as the model fish. The mutant gene may have a deletion mutation that causes a deletion of at least a part of the protein encoded by the PKD2 gene. Furthermore, the mutant gene may have a deletion mutation that causes a C-terminal deletion of the protein encoded by the PKD2 gene.

 According to the present invention, there is provided a screening method for an external factor related to the onset or progress of human polycystic kidneys, the method comprising providing an external factor to be screened to any of the above polycystic kidney model small fishes; And a step of evaluating a promoting action or an inhibitory action on the onset and / or progression of human polycystic kidney in the model fish to which the external factor has been imparted.

 In the present screening method, the external factor can be a compound or an environmental factor other than the compound. Further, the external factor is a compound, and the evaluation step can be a step of evaluating an action of suppressing the onset or progress of the human polycystic kidney disease.

 The polycystic kidney model fish of the present invention is a fish obtained by introducing a dominant negative mutant gene of the PKD2 gene. According to the fish of the present invention, it is possible to obtain a transgenic small-sized fish that causes a large number of cysts and is not embryonic lethal due to mutations in the gene responsible for human ADPKD. That is, it is possible to obtain a model fish having the onset and progress of human ADPKD. Moreover, according to the small fish of this invention, since it is a genetically modified fish, systematization can provide a fish. Furthermore, according to the small fish of the present invention, external factors related to human ADPKD can be screened easily and rapidly at low cost from the characteristics of the small fish.

 In addition, the screening method for external factors related to the onset or development of human polycystic kidneys of the present invention provides the external factors to be screened to the fish of the present invention, and multiple cysts in model fish to which the external factors have been added. It is a method for evaluating a promoting action or an inhibitory action on the onset and / or development of the kidney. According to this screening method, an external factor related to the onset of human ADPKD and the like can be screened appropriately, simply and rapidly. It can also be used to elucidate the pathogenesis of human ADPKD.

 Hereinafter, embodiments of the polycystic kidney model small fish of the present invention and use thereof will be described in detail.

(Multiple cyst kidney model small fish)
The polycystic kidney model small fish of the present invention (hereinafter also simply referred to as the present small fish) is a genetically modified small size obtained by introducing a mutant gene of the PKD2 gene so that a dominant negative effect can be expressed by genetic recombination. It is a fish.

(Small fish)
The small fish for obtaining the present small fish is preferably a small fish for research or experimental use, more preferably a small fish of teleost, such as zebrafish, medaka, goldfish, loach, tiger trout, etc. . Among these, transparent small fish that can visually recognize internal organs from outside the body is preferable. As such a transparent small fish, for example, a small fish belonging to the genus genus including the transparent medaka disclosed in JP-A-2001-328480 or the like or a related fish can be preferably used. Also, zebrafish can be preferably used.

(Transgene)
The dominant negative mutant gene to be introduced may be a mutant of the PKD2 gene derived from a small fish of the same or different species as the small fish to be genetically modified, or may be a mutant of the PKD2 gene of a heterologous animal. It may be. The small fish PKD2 gene is often highly homologous to the gene of a mammal such as a human, and therefore the PKD2 gene of the small fish may not necessarily be mutated.

 The PKD2 gene is already known in humans (accession number: NM_000297), mice (accession number: NM_008861), and zebrafish (accession number: NM_001002310). The PKD2 gene of animals including small fish that is not currently known is the nucleotide sequence or amino acid sequence of the known small fish PKD2 gene and / or the known nucleotide sequence or amino acid sequence of the PKD2 gene of a different animal such as a human or mouse. Based on the above, homologous search using publicly available databases (National Center for Biotechnology Information, USA and Medaka genome project by NIG DNA Sequencing Center, Japan) regarding gene base sequences and amino acid sequences Can be obtained at. For example, the medaka PKD2 gene was first identified by the present inventors, and the puffer PKD2 gene was predicted on the puffer database (DOE Joint Genome Institute, USA) based on the mouse PKD2 gene. The homologous search was conducted in the medaka database to obtain the medaka PKD2 gene (SEQ ID NO: 3). The medaka PKD2 gene showed relatively high homology with human and mouse PKD2.

 The PKD1 gene, which is another causative gene, is already known in humans (accession number: NM_001009944) and mice (accession number: NM_013630. Animals including small fish that are not currently known. The PKD1 gene is based on the nucleotide sequence or amino acid sequence of the known small fish PKD1 gene and / or the known nucleotide sequence or amino acid sequence of the PKD1 gene of a different animal such as human or mouse. It can be obtained by performing a homologous search using publicly available databases (National Center for Biotechnology Information, USA and Medaka genome project by NIG DNA Sequencing Center, Japan), etc. For example, medaka The PKD1 gene was first identified by the present inventors, but the amino acid level was determined based on the base sequence of the puffer PKD1 gene. Belle homologous medaka sequences have been obtained by BLAST search on the medaka database (Medaka genome project by NIG DNA Sequencing Center, Japan) (SEQ ID NO: 1) The medaka PKD1 gene is the pufferfish PKD1 gene. However, the homology with the PKD1 gene of mammals such as humans and mice is low.

 The transgene is preferably one that causes a dominant negative effect to be expressed by gene recombination that introduces the mutated gene by mutating the PKD2 gene. If the mutation in the PKD2 gene can express a dominant negative effect, the expression of the dominant negative effect can be controlled by adjusting the introduction amount, expression time, and expression level of the mutant gene.

 The mutation for the gene can be one or a combination of two or more selected from base substitution, deletion, insertion and addition, and the number of bases related to such mutation is not particularly limited. In addition to mutations that cause substitution, deletion, insertion, and addition of one or more amino acid residues in the amino acid sequence of the product of the PKD2 gene, mutations include forms of nonsense mutations, nonsense mutations, and frameshift mutations. Can be taken.

 Examples of dominant negative mutations in the PKD2 gene include deletion mutations that lack the C-terminal side of the gene product PC2. The deletion on the C-terminal side only needs to be deleted to such an extent that a dominant negative effect can be effectively expressed. For example, the human PKD2 gene and the medaka PKD2 gene are both divided into 15 exons. It can be a mutation that deletes all or part of 12-15. Preferably, it is set as the mutation which deletes all the exons 12-15. The dominant negative mutation of the PKD2 gene of other small fish or animals is preferably a mutation that deletes all or part of the region corresponding to exons 12 to 15 of the medaka or human PKD2 gene.

 Such a mutant gene, for example, a DNA comprising a base sequence in which one or several bases have been deleted, substituted or added, can be any known to those skilled in the art such as chemical synthesis, genetic engineering techniques, mutagenesis, etc. It can produce by the method of.

 In addition, the mutant form of the PKD2 gene that allows the dominant negative effect to be expressed by mutating the PKD2 gene is not limited to this, and small fish that have been prepared by various PKD2 mutant genes and genetically modified as transgenes. By evaluating whether or not the function of the normal PKD2 gene product is inhibited by using as an index the form of cyst kidney formation in polycystic kidneys, an effective mutant form of the PKD2 gene can be obtained. In this evaluation, for example, a technique for observing a cyst in a tissue section of the obtained transgenic small fish can be employed.

(DNA construct for mutated gene introduction)
In order to produce transgenic small fish using the PKD2 mutant gene, it is necessary to introduce the mutant gene product as a DNA construct that can be expressed in small fish cells. The combination of elements for gene expression in such a DNA construct and the sequence thereof are not particularly limited, but a necessary regulatory region such as a polyadenylation sequence is added in addition to linking a PKD2 mutant gene under the control of an appropriate promoter. be able to. In addition, an intron sequence, an enhancer sequence having a function of enhancing gene expression, or a terminator sequence that directs transcription termination can be used as necessary.

 The promoter is not particularly limited as long as it can function in small fish. It may be a constitutive promoter or an inducible promoter. Whether to use a constitutive promoter or an inducible promoter can also be determined by the degree of dominant negative effect obtained by the mutated gene. For example, when the degree of functional suppression of the normal PKD2 gene is strong, an effective human ADPKD model may be established by controlling the expression intensity using an inducible promoter. In addition, when the degree of function suppression of the normal PKD2 gene of the mutant gene is moderate, it may be established as an effective human ADPKD model by expressing it using a constitutive promoter.

 As the inducible promoter, various commercially available systems can be used. An example is Tet-On and Tet-Off gene expression systems (Takara Bio) that can be induced by doxycycline. Furthermore, in the case of a PKD2 mutant gene, it may be expressed as a fusion protein of a mutant protein of PKD2 and various reporter proteins so that the expression state can be easily confirmed. Examples of reporter protein genes include fluorescent protein genes such as GFP, lacZ gene, soluble alkaline phosphatase gene, and luciferase gene.

 Moreover, a promoter with high tissue specificity may be sufficient, and a promoter with low tissue specificity may be sufficient. Considering tissue specificity, it is necessary to be a promoter capable of expressing a PKD2 mutant gene in at least the kidney. Considering that human ADPKD is inherently a systemic disease and a disease affecting many types of organs, it is preferable to use a promoter expressed in tissues other than muscle. Examples of such a promoter include EF-1α-A promoter and β-actin promoter.

 The form of the DNA construct is not particularly limited. As will be described later, it can be appropriately selected according to the gene transfer mode for fertilized eggs and the like. The DNA construct may be naked or may be in the form of a vector held on a suitable plasmid.

 The transgenic small fish can be produced based on a known method for producing a transgenic small fish, such as introducing a DNA construct for gene introduction into a fertilized egg of a small fish. That is, it can be performed by a microinjection method, viral vector infection method, particle gun method, or electroporation method to an oocyte or a fertilized egg.

 The small fish includes adult fish into which such a mutated gene has been introduced, its progeny, fish fertilized egg cells, fish embryo cells, and the like.

(Screening method for external factors related to the onset or progression of human polycystic kidney disease)
The screening method of the present invention comprises a step of applying an external factor to be screened to the small fish, and an action of promoting or suppressing the onset and / or progression of multiple cystic kidneys in the small fish to which the external factor is applied. A process of evaluating;
Can be provided.

(External factor application process)
The external factor to be screened is not particularly limited. It may be related to the onset and progression of human ADPKD. For example, the external factor may be a compound or an environmental factor that can be used as a drug for suppressing or preventing human ADPKD, that is, a preventive or therapeutic agent, It may be an environmental factor such as stress. The external factor to be imparted may be not only one kind but also plural kinds. For example, it may be both a compound and environmental stress, or two or more kinds of compounds that may have a synergistic effect. Two or more types of compounds that may have a canceling action may be used. Since this screening method can prepare a large amount of model animals, screening combining two or more external factors can be performed without incurring a large cost.

 In order to impart an external factor to the small fish, if the small fish is a larvae or an adult fish, an external factor such as a compound may be included in the feed of the larvae or the adult fish. The compound may be supplied or various environmental factors such as temperature may be imparted. Moreover, an external factor can also be included in the gas containing oxygen supplied to the breeding liquid. When this small fish is in the process of development, the medium can add compounds and environmental factors to the gas being cultured.

(Evaluation process)
There is no particular limitation on the method for evaluating the promoting and inhibiting effects on the onset and / or progression of multiple cystic kidneys in this small fish. Examples of such an index for evaluation include the state of the present small fish during development or rearing, the state of a cyst in a tissue section, and the like. In addition, it may be anything as long as it accompanies an increase or decrease in cysts. For example, it may be a new index detected from the composition in the breeding liquid at the time of screening, or a new index detected from the small fish at the time of screening. In other words, in addition to the external factor process and the evaluation process, it is possible to suppress the onset and / or progression of human APKD by performing the process of evaluating the presence or absence of factors that increase or decrease in the rearing liquid and the small fish being bred. Or new indicators can be found that can monitor the promotion.

 In the evaluation process, in order to evaluate the promoting and / or inhibiting effects on the onset and / or progression of multiple cystic kidneys, this small fish is reared under conditions such as rearing that can be used as a control when no external factor is applied. Alternatively, data as such a control can be acquired in advance and compared with the tendency of the onset and progression of multiple cystic kidneys in the control.

 In the evaluation process, if the external factor is positive for the effect on the onset and / or progression of multiple cystic kidneys in this small fish, the external factor is an external factor that adversely affects the onset and / or progression of human ADPKD. Is a factor. Therefore, from the viewpoint of prevention / treatment of human ADPKD, another external factor capable of inactivating, degrading or removing such an external factor is newly screened. For example, in this screening method, there is a possibility that an external factor that has an adverse effect is previously given to the breeding environment such as liquid for breeding the small fish or feed, and the influence of this external factor is mitigated or neutralized. What is necessary is just to give a certain external factor and to evaluate the effect | action.

 In addition, in the evaluation process, when the inhibitory action on the onset and / or progression of multiple cystic kidneys in the small fish is affirmed with respect to the external factor, the external factor is used to prevent the onset and / or progression of human ADPKD or It is an external factor that favors treatment. Such an external factor can be used as a drug for the prevention / treatment of human ADPKD, and can also be used as an environmental factor preferable for the prevention / treatment of human ADPKD.

 Hereinafter, the present invention will be specifically described with reference to examples. However, this example does not limit the present invention.

(Cloning of medaka Pkd1 and Pkd2)
Databases (National Center for Biotechnology Information, USA and Medaka genome project by NIG DNA) to identify medaka genome sequences that are homologous at the amino acid level with the base sequence of pufferfish Pkd1 (Hum Mol Genet 1997; 6: 1483-1489.) Blast search was performed on Sequencing Center, Japan. As for medaka Pkd2, first, puffer Pkd2 was predicted on the puffer database (DOE Joint Genome Institute, USA) based on mouse Pkd2, and homologous search was performed based on this.

  In the obtained medaka Pkd1 and Pkd2, RT-PCR was performed by setting a primer at a place presumed to be an exon. As an RT-PCR template, an egg and kidney cDNA library prepared from wild type medaka OR (orange red) by the Marathon method (Marathon cDNA Amplification Kit; Takara Bio, Otsu, Shiga, Japan) was used. For both ends of the gene, 5′RACE and 3′RACE methods were used according to the protocol attached to the kit. The obtained PCR product was subcloned into a vector and subjected to sequence analysis. The results are shown in FIG. 1 and Tables 1 and 2.

(1) Medaka Pkd1
As shown in FIG. 1A and Table 1, the gene size was approximately 80 kbp, and the mRNA translation region was 13791 bp and was divided into 56 exons (FIGS. 1A to 1N). The underlined sites in the base sequences of FIGS. 1B to 1Q are each the last base of the exon, and the numbers described in the right column of the base sequence indicate the numbers of the exons including the last base. . The homology at the amino acid level was 49.6% for human PKD1, 48.4% for mouse Pkd1, and 68.7% for puffer fish Pkd1. A long intron 1 found in other species was also found in medaka Pkd1. Puffer exon 2 and exon 31 were each divided into two in Medaka, and as a result, Medaka Pkd1 consisted of 56 exons. Table 1 shows a comparison of human, puffer fish, and medaka Pkd1, and shows the correspondence between the domains on the human PKD1 database (NCBI) and the exons of each species.

(2) Medaka Pkd2
As shown in FIG. 1A and Table 2, the gene size was approximately 9.2 kbp, and the translation region of mRNA was 2706 bp and was divided into 15 exons (FIG. 1A, FIG. 1O to FIG. 1Q). Amino acid homology was 64.6% for human PKD2 and 65.2% for mouse Pkd2. Like human PKD2, it was divided into 15 exons. Table 2 shows a comparison between human and medaka Pkd2, and shows the correspondence between the domains on the human PKD2 database (NCBI) and the exons of each species.

 Medaka Pkd1 showed high homology with pufferfish Pkd1, but low homology with mammals such as humans and mice. In contrast, medaka Pkd2 showed relatively high homology with human and mouse PKD2. The presence of the PKD2 homologous gene in C. elegans [22] suggests that it is well conserved across species during evolution and plays an extremely important function for organisms.

 In this example, various constructs shown in FIG. 2 were prepared, and a transient expression experiment was performed by gene introduction by a microinjection method.

(Deletion mutant construct production)
Various constructs shown in FIG. 2 were prepared. In medaka Pkd1, the plasmid construct EF-1α-A-EGFP-Pkd1ΔN was deleted from exon 6 to exon 27 to replace the N-terminal other than the signal peptide and replaced with enhanced green fluorescent protein EGFP (Takara Bio). Created.

 In the medaka Pkd2, a plasmid construct EF-1α-A-Pkd2ΔC-EGFP was prepared by adding EGFP as a tag to the 3 ′ side of the mutant from which the C-terminal from exon 12 to exon 15 was deleted. As a promoter, an EF-1α-A promoter (Develop Growth Differ 2000; 42: 469-478.) Expressed in the whole body other than muscle was used.

 In addition, in order to create a strain using the Tet-On / Off induction system, a commercially available Tet-On and Tet-Off gene expression systems (Takara Bio) was used to convert the transcription factor rtTA for Tet-On to EF-1α- A construct EF-1α-A-tTA that expresses a construct EF-1α-A-rtTA expressed by the A promoter and a transcription factor tTA for Tet-Off was prepared. Constructs TRE2-EGFP-Pkd1ΔN and TRE2-Pkd2ΔC-EGFP having a tet response element corresponding to this and having a response gene whose expression is regulated by doxycycline were also prepared.

(Gene transfer by micro-injection method)
Using a micromanipulator and a micrometer syringe in the fertilized egg cytoplasm of the 1 to 4 cell stage of wild type medaka OR (orange red), the gene was introduced under the microscope by the micro-injection method [21]. The DNA concentration was adjusted to 25 ng / μl.

(result)
(1) Transient expression experiment of EGFP-Pkd1ΔN
Plasmid construct EF-1α-A-EGFP-Pkd1ΔN was introduced into 275 fertilized eggs by the micro-injection method. The results are shown in Table 3 and FIG. After 24 hours, 104 eggs fluoresced with EGFP. Heart inversion was observed in one egg. Thirty-five hatched and 12 animals had trunk flexion. As a result of evaluation by tissue section, cysts of the pronephros were observed in 1 out of 25 animals (FIGS. 3A and 3B).

Table 3 shows the results of transient expression experiments. That is, it shows a list of phenotypes recognized in fertilized eggs and fry after gene transfer by the micro-injection method. However, fluorescence; 24 hours after micro-injection, the number of eggs that exhibited green fluorescence by EGFP, heart inversion; the number of individuals found to have heart inversion by observation of fertilized eggs on the 6th day, flexion; hatching Number of juveniles with trunk flexion, straight; Number of juveniles hatched without abnormalities, slices; Number of mainly bent juveniles fixed and tissue sections prepared; kidney cysts; Prosthetic cysts confirmed by microscopic examination of tissue sections Number of individuals.

(2) Transient expression experiment of Pkd2ΔC-EGFP
Plasmid construct EF-1α-A-Pkd2ΔC-EGFP was introduced into 452 eggs by micro-injection method. The results are shown in Table 3 and FIG. After 24 hours, 135 eggs fluoresced with EGFP. When the eggs on the 6th day after fertilization immediately before hatching were observed with a stereomicroscope, heart inversion was observed in 8 eggs (FIGS. 4C and D). 74 hatched and 29 animals showed trunk flexion (FIG. 4A). Evaluation by histological section revealed prorenal cysts in 3 of 24 animals (FIG. 4B).

 In medaka, transgenes can be expressed in vivo by micro-injecting plasmid constructs into the cytoplasm of the 1st to 4th cell stages of fertilized eggs. The transgenic egg hatched in about 7 days, and the fry had all the basic organs of the vertebrate, so it was possible to easily evaluate the function of the transgene. In this experiment, the mutant medaka Pkd2 with the C-terminal deleted by the EF-1α-A promoter, which is a peptide chain elongation factor promoter, was overexpressed at the early embryonic stage. As a result, it was considered that a cyst was formed in the pronephros, and the deletion mutant protein worked as a dominant negative mutant, resulting in a phenotype equivalent to that of the knockout animal. In addition to renal cysts, cardiac inversion and flexion were also recognized as transient deletion mutant gene expression phenotypes. About 0.5% of wild-type medakas have cardiac inversions, but in transient expression experiments, 6% of eggs that were micro-injected with Pkd2 deletion mutants and showed fluorescence showed heart inversions. . Human ADPKD does not show heart inversion, but Pkd2 knockout mouse homozygotes show heart inversion in 33%. This is because polycystin-2 is involved in the determination of the left and right axis in early embryos, and it is thought that cardiac inversion occurs when its function is inhibited. Therefore, the inversion of the heart was observed in the early embryos of the medaka that was transiently expressed but introduced the mutant Pkd2. The possibility that the Pkd2 deletion mutant inhibited wild-type Pkd2 through a dominant negative mechanism. Was strongly suggested.

 Stem flexion is observed in 0.5% of hatched fry in the wild type, but in 13% of eggs that showed fluorescence when the Pkd1 deletion mutant was introduced, and 33% when the Pkd2 deletion mutant was introduced. Admitted. Although it was considered that bending could occur due to the effect of the micro-injection itself, renal cysts were observed only in bent fry in this experiment. In zebrafish and many zebrafish mutants with kidney cysts that have knocked down Invs or Pkd2 gene expression by Morpholino anti-sense oligonucleotide, it has been reported that flexion appears as a phenotype along with kidney cysts. The relationship between renal cyst and flexion was suggested.

 Another thing to note is that more than half of them died without hatching after micro-injection. Most of the homozygotes of Pkd1 and Pkd2 knockout mice are fetally lethal, but have multiple cystic kidneys in which almost no renal parenchyma is observed in the embryonic period. It was also possible that the deletion mutant-introduced medaka could not be hatched due to embryonic lethality when the mutant was overexpressed during embryogenesis, but a kidney cyst was formed. The medaka that had escaped embryonic lethality and was hatched would rather have insufficient mutant mutant gene expression and less cyst formation.

 In this way, gene transfer of Pkd1 and Pkd2 deletion mutants may cause bending and lethality when a sufficient amount of kidney cysts is introduced, and systematic transgenic medaka that can regulate some expression. Was considered necessary. For this reason, in addition to the strain that expresses the transgene constitutively by the EF-1α-A promoter, a strain that regulates gene expression by administering doxycycline using the Tet-On / Off induction system is also created, Further study was conducted.

(Establishment of transgenic medaka lines)
In this example, from an adult fish obtained from a transient expression experiment, an individual into which a transgene was introduced by a PCR method was identified, and this individual was crossed with a wild type, and an individual in which gene introduction was further confirmed. F1 generation was acquired by crossing with the wild type, and F2 generation was further acquired.

(Systematization and genotyping)
After larvae hatched from eggs after micro-injection were raised for 2-3 months and became adults, DNA was extracted from the tail fin by the following method, and the transgene was introduced by the PCR method shown in Table 4 below. Identified individuals. Table 4 shows PCR primers (SEQ ID NO: 5 to SEQ ID NO: 14) used for genotyping. The combination of mep1-B1F and GFP3 also detects the EF-1α-A-EGFP-Pkd1ΔN line. The P2HindF and GFP3 pair also detects the EF-1α-A-Pkd2ΔC-EGFP strain.

 The tail fin of an adult medaka fish was placed in 50 μl of lysis buffer (10 mM Tris-HCl, 50 mM KCl, 0.3% Tween20, 1 mM EDTA). After heating at 95 ° C. for 10 minutes, 2.5 μl of Proteinase K (20 mg / ml) was added and heated at 55 ° C. overnight. The next day, the mixture was heated at 95 ° C. for 10 minutes and diluted 5-fold with distilled water. 1 μl of this was used for PCR.

 Since the fish has a mosaic gene integration, this individual was bred with the wild type, and the individual into which the transgene was introduced into the germ cells was further identified by PCR using the DNA of the fertilized egg. The F1 generation obtained by crossing this individual with the wild type became an individual in which the transgene was integrated into all cells. In fish, it is known that the insertion site and copy number of the transgene may be different for each individual even in the F1 generation, and one animal was selected from the F1 generation and mated with the wild type. As a result, the F2 generation and subsequent generations have inherited patterns according to Mendel's law, resulting in the establishment of strains. The results are shown in Table 5.

Table 5 shows a list of the number of transgenic medakas that have been transfected by the micro-injection method and the transgene has been incorporated into germ cells. However, somatic cell gene positive; number of individuals positive for transgene by genotyping using tail fin, germline gene positive; and mating of these individuals with wild type, and positive genotyping of the resulting fertilized egg It was the number of individuals.

(result)
As shown in Table 5, the Tet-On / Off induction system was used to create a strain that can be bent and lethal in gene transfer of a deletion mutant and can regulate expression. Tet-On / Off strains were obtained by establishing a strain with a transcription factor and a strain with an induced gene, respectively, and then mating them. One EF-1α-A-rtTA strain as a medaka with the transcription factor rtTA used in the Tet-On induction system, and EF-1α-A- as a strain with the transcription factor tTA for the Tet-Off induction system Three tTA lines were established.

(1) System with Pkd1ΔN
Three TRE2-EGFP-Pkd1ΔN strains were created as medakas with Tet-On / Off induction system response genes. The no.7 strain with the highest copy number was selected from the Southern blot results and crossed with the EF-1α-A-tTA no.6 strain to obtain a Tet-Off EGFP-Pkd1ΔN strain. Furthermore, two lines of EF-1α-A-EGFP-Pkd1ΔN were obtained as lines expressing the transgene without involving the induction system.

(2) System with Pkd2ΔC
Two strains of TRE2-Pkd2ΔC-EGFP were created as medakas with Tet-On / Off induction system response genes. The no.17 strain with the highest copy number was selected from the Southern blot results and crossed with the EF-1α-A-rtTA strain to obtain the Tet-On Pkd2ΔC-EGFP strain. Five strains of EF-1α-A-Pkd2ΔC-EGFP were obtained as constitutively expressing transgenes.

(Transgene confirmation by Southern blotting and copy number estimation)
In this example, genomes were extracted from the systematized individuals, and it was confirmed by the DIG-Southern blotting method that the full length of the transgene was integrated, and the copy number was estimated. As a positive control, the plasmid construct used to make each strain was used with the concentration adjusted.

(Genome extraction)
All medaka adult fish were placed in 5 ml of lysis buffer (50 mM Tris-HCl, 20 mM EDTA, 100 mM NaCl) and homogenized. Thereafter, 250 μl of 10% SDS and 125 μl of Proteinase K (20 mg / ml) were added and heated at 55 ° C. overnight. The next day, the mixture was centrifuged at 3000 rpm and room temperature for 5 minutes, and only the supernatant was transferred to another tube. 1.25 ml of 5M NaCl was added and placed on ice for 15 minutes. The mixture was centrifuged at 3000 rpm and room temperature for 5 minutes, the supernatant was transferred to another tube, 2 ml of distilled water was added, and the mixture was centrifuged again at 3000 rpm and room temperature for 15 minutes. Only the supernatant was transferred to another tube, and 0.8 times the volume of isopropanol was added to form a DNA string-like precipitate. The precipitate was transferred to an Eppendorf tube, rinsed with 70% ethanol, dissolved in 200 μl of TE, added with 2 μl of RNase (10 mg / ml), and heated at 37 ° C. for 1 hour. After phenol / chloroform treatment twice, ethanol precipitation was performed and dissolved in 200 μl of TE.

(Southern blotting (DIG method))
The genome was extracted from all medaka individuals by the above method, and 8 μg was used for each. In the line with EGFP-Pkd1ΔN, the genome was cut with EcoRI / BglII, and the line with Pkd2ΔC-EGFP was cut with BamHI. The probe labeled with DIG-dUTP (Roche, Basel, Basel-Stadt, Switzerland) against medaka Pkd1 is a primer 5'-GCTCATCCTCCACCAGTGAC-3 'using the construct EF-1α-A-EGFP-Pkd1ΔN as a template targeting exon 56 (SEQ ID NO: 15) and 5′-CCCTCCCTCTCTGAAAGGTT-3 ′ (SEQ ID NO: 16) were used to prepare the PCR method. The probe for medaka Pkd2 is the primer 5'-CTGATGTTTTGCGCTTTGTC-3 '(SEQ ID NO: 17) and 5'-GGGATGCTCGTGTCTGTCTT-3' (SEQ ID NO: 18) using the construct EF-1α-A-Pkd2ΔC-EGFP as a template for exon 1. The PCR method was used.

 Transfer to a positively charged nylon membrane (Hybond N +; GE healthcare Bio-Science Corp, Piscataway, New Jersey, USA) by an alkaline transfer method according to a standard protocol. CDP-star (Roche) was used as a chemiluminescent substrate, and development was performed with a CCD camera (Cool saver; Atou, Tokyo, Japan). The results are shown in FIG.

(result)
(1) System with Pkd1ΔN
Southern blotting of TRE2-EGFP-Pkd1ΔN line and EF-1α-A-EGFP-Pkd1ΔN line was performed. In calculation, 100 pg of plasmid corresponded to 1 copy. The highest number of copies in each strain is approximately 10 copies of the EF-1α-A-EGFP-Pkd1ΔN no.1 strain (FIG. 5A), and approximately 10 copies of the TRE2-EGFP-Pkd1ΔN no.7 strain ( Figure 5B).

(2) System with Pkd2ΔC
Southern blotting of TRE2-Pkd2ΔC-EGFP strain and EF-1α-A-Pkd2ΔC-EGFP strain was performed. Both TRE2-Pkd2ΔC-EGFP no.17 and no.24 lines were estimated to be less than 10 copies (FIG. 5C). Among the EF-1α-A-Pkd2ΔC-EGFP lines, the no.7 and no.12 lines had 10 copies or more, and it was considered that there were many copy numbers (FIG. 5D).

(Confirmation of expression by RT-PCR)
In this example, it was confirmed by RT-PCR that the transgene integrated in the genome was expressed using primers specific to the transgene.

 Medaka liver and kidney were excised and stored in RNAlater (QIAGEN, Hilden, North Rhine-Westphalia, Germany). Total RNA was extracted using RNeasy mini kit (QIAGEN) by adding RNase-free DNase I (QIAGEN) treatment on a silica membrane according to the protocol attached to the kit. Further, DNase I (Invitrogen, Carlsbad, California, USA) treatment was performed, and after phenol / chloroform treatment and ethanol precipitation, the RNA concentration was adjusted to 1 μg / μl. Using 1 μl of this total RNA as a template, cDNA was obtained by reverse transcription with SuperScriptIII Reverse Transcriptase (Invitrogen) using a random hexamer. After RNase (SIGMA, Saint Louis, Missouri, USA) treatment, using this cDNA as a template, for expression of Pkd1ΔN, mep-1B1F primer and GFP3 primer (Table 4) were used, and Pkd2ΔC was P2HindF primer and GFP3 primer PCR reaction was performed using (Table 4). As a negative control, PCR reaction was performed under the same conditions using RNA before reverse transcription. As PCR conditions, Ex Taq (Takara Bio) was used, and heat denaturation 94 ° C., 30 seconds, annealing temperature 60 ° C., 30 seconds, extension reaction 72 ° C., 1 minute, 35 cycles. The results are shown in FIG.

(result)
(1) System with Pkd1ΔN
In the EF-1α-A-EGFP-Pkd1ΔN no.1 liver and kidney RT-PCR, a target 730 bp band was observed only when reverse transcriptase was added (FIG. 6A).
(2) System with Pkd2ΔC
In the EF-1α-A-Pkd2ΔC-EGFP no.12 liver and kidney RT-PCR, a target 657 bp band was observed only when reverse transcriptase was added (FIG. 6B).

(Confirmation of transcription factors rtTA and tTA by Western blotting)
Medaka liver was homogenized in RIPA buffer and electrophoresed using a 10% SDS PAGE gel. Blotting was performed on PVDF membrane (Immobilon-P; Millipore, Billerica, Massachusetts, USA) with a wet type blotting apparatus. Anti-VP16 antibody (SIGMA) was diluted 5000 times as the primary antibody, and HRP-Goat Anti-Rabbit IgG antibody (H + L) (Zymed, San Francisco, California, USA) was diluted 20000 times as the secondary antibody. Immobilon Western (Millipore) was used as a chemiluminescent substrate and developed with a CCD camera (Cool saver; Ato). The results are shown in FIG.

 The target protein of 37 kDa in the EF-1α-A-rtTA and EF-1α-A-tTA no.6 strains by Western blotting using an antibody against the VP16 transcription active domain in the transcription factors rtTA and tTA (anti-VP16 antibody) Was confirmed to be expressed (FIGS. 7A and 7B).

(Tissue section of a transgenic medaka)
In this example, the juvenile fish and adult fish were fixed in bud and then embedded in paraffin. Serial sections were prepared using a microtome, and HE staining was performed.

 Transgenic medaka was bred, tissue sections were periodically prepared, and renal cysts were searched. The results are shown in FIGS.

(result)
(1) Tissue search of adult EF-1α-A-EGFP-Pkd1ΔN no.1 heterozygous fish, which was thought to have a large number of copies with Pkd1ΔN, showed no renal cysts at 5 months . The Tet-Off EGFP-Pkd1ΔN line should express the transgene with maximum activity without doxycycline, but no kidney cysts were found in tissue sections of adult fish 6 months later.

 From the above, the medaka Pkd1ΔN mutation can be summarized as follows. That is, when an N-terminal deletion mutant of medaka Pkd1 was introduced into a fertilized egg by the micro-injection method, kidney cysts and trunk trunk flexion were observed in the fry. N-terminal deletion mutant of medaka Pkd1 was introduced and systematized, but no renal cyst was observed.

(2) System with Pkd2ΔC
After breeding EF-1α-A-Pkd2ΔC-EGFP no.12 line for 7 months, a tissue section of heterozygote was prepared and microscopically examined. Several moderate renal cysts were found in both kidneys (FIGS. 8A, 8B, 8C, 8D). In addition, after exposing the Tet-On Pkd2ΔC-EGFP strain to doxycycline at 10 μg / ml for 4 months, a microscopic examination of a tissue section of an individual in which both the transcription factor rtTA and the response gene TRE2-Pkd2ΔC-EGFP are heterozygotes was performed. Several renal cysts were found in the kidney (FIGS. 9A, 9B, 9C).

 Based on the above, the medaka Pkd2ΔC mutation can be summarized as follows. In other words, when a C-terminal deletion mutant of medaka Pkd2 was introduced into fertilized eggs using the micro-injection method, kidney cysts, heart inversion, and trunk flexion were observed in the fry. In addition, a transgenic medaka that forms a renal cyst could be systematized using a medaka Pkd2 C-terminal deletion mutant.

(Summary)
In medaka, the transgene is integrated in a mosaic pattern, so the probability of entering a germ cell is as low as 10%. However, since egg collection is easy, it is possible to micro-injection into a large number of fertilized eggs, and it is possible to produce transgenic medakas more easily than mice as a whole if they are proficient in the technique. Transgenic medakas that are systematized express the transgene in all cells. However, the expression level of the gene integrated into the genome is reduced compared to the transient experiment. Only a few percent of the micro-injected transgene is integrated into the genome, and its expression is also affected by the insertion position on the genome. In this experiment as well, in the transient expression experiment, the fluorescence of EGFP used as a tag could be observed under a stereomicroscope, but it was impossible to visually confirm the fluorescence with a systematic transgenic medaka. This was thought to be due to the difference in the expression level.

 A histological study of systematic transgenic medaka was performed. In the EF-1α-A-EGFP-Pkd1ΔN line introduced with the Pkd1 N-terminal deletion mutant, no medaka that forms a renal cyst was observed. The medaka in which the deletion mutant was expressed to the extent that a kidney cyst was formed was lethal. Therefore, a line with the transcription factor tTA and a line with the response gene TRE2-EGFP-Pkd1ΔN were created, and these two lines were crossed to express the N-terminal deletion mutant of Pkd1 by the Tet-Off induction system. However, similar to the EF-1α-A-EGFP-Pkd1ΔN strain, phenotypes such as renal cysts and flexion were not observed. Pkd1 has been shown to form renal cysts by micro-injection and overexpression of human PKD1 C-terminal mRNA in eggs in zebrafish [25] [26]. The phenotype observed in the experiment was not due to the inhibition of wild-type Pkd1 due to the dominant negative effect, but could be due to overexpression of the C-terminus. In any case, it was speculated that the expression level of the deletion mutant was not sufficient in the systematic Pkd1 mutant medaka and cyst formation was not observed.

 On the other hand, the Tet-On Pkd2ΔC-EGFP line using the Tet-On induction system continued to be exposed to doxycycline at a concentration of 10 μg / ml, and individuals that formed renal cysts appeared 4 months later. The EF-1α-A-Pkd2ΔC-EGFP strain that constitutively expressed the transgene could also be systematized without being lethal due to gene expression, contrary to the original hypothesis. The no.12 line, which was considered to have the highest copy number, formed a renal cyst after 7 months, but the number of renal cysts was small. The other low-copy-number lines did not form renal cysts even after 7 months, and sufficient expression was considered necessary for the deletion mutant to function as a dominant negative mutant. In transient experiments in which a large amount of mutants were expressed, renal cysts were observed at the stage of fry, whereas in medakas that were systematized, it took several months to form cysts. It was assumed that the smaller the amount, the longer the time required for cyst formation. Neither trunk flexion nor cardiac inversion was observed in both strains. This is rather convenient as a model for forming only a renal cyst. Increasing the expression level of deletion mutants to the extent that trunk flexion or cardiac inversion is not observed is expected to cause early cysts and an increased frequency of cysts, depending on the phenotype of human ADPKD. Expected to approach.

 From the above, Pkd1 N-terminal deletion mutant-introduced transgenic medaka did not obtain a phenotype, but Pkd2 C-terminal deletion mutant-introduced transgenic medaka had a dominant negative mutant with sufficient expression. It was found that it can function as a renal cyst. It was also found that transgenic medaka transgenic for Pkd2 deletion mutant is a new model animal that is effective in drug exposure tests for cyst formation.

Sequence number 5-18: Primer

It is a schematic diagram of the structure of the medaka Pkd gene. A shows that it is divided into 1.56 exons of medaka Pkd. The parenthesis indicates the exon number of the corresponding pufferfish, and B indicates that it is divided into medaka Pkd 2.15 exons. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD1 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD2 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD2 gene. It is a figure which shows a part of cDNA sequence and amino acid sequence of the medaka PKD2 gene. It is a construct schematic diagram. The arrow (→ ←) on each construct indicates the approximate position of the PCR primer for detection (Table 1). A) The plasmid construct EF-1α-A-Pkd2ΔC-EGFP incorporated cDNA fused with EGFP by deleting the C-terminus of exon 12 of medaka Pkd2 downstream of the EF-1α-A promoter. BamHI indicates a restriction enzyme site. B) Plasmid construct EF-1α-A-EGFP-Pkd1ΔN deletes the N-terminal other than exons 1 to 5 containing the signal peptide of medaka Pkd1 downstream of the EF-1α-A promoter, and fused EGFP instead. Incorporated cDNA was incorporated. EcoRI and BglII indicate restriction enzyme sites. C) The plasmid construct EF-1α-A-rtTA incorporated a transcriptional activator rtTA for Tet-On downstream of the EF-1α-A promoter. D) Plasmid construct TRE2-Pkd2ΔC-EGFP incorporated a cDNA lacking the C-terminus of medaka Pkd2 downstream of the minimized CMV promoter with tetracycline response factor (TRE). E) In the plasmid construct TRE2-EGFP-Pkd1ΔN, a cDNA lacking the N-terminal other than the signal peptide of medaka Pkd1 was incorporated downstream of the minimized CMV promoter having a tetracycline response factor (TRE). F) The plasmid construct EF-1α-A-tTA incorporated a transcriptional activator tTA for Tet-Off downstream of the EF-1α-A promoter. The figure which shows the fry which transfected EGFP-Pkd1ΔN transiently. A) Appearance. Acknowledging trunk flexion. B) A section of the fry two days after hatching. Dilation (*) of Bowman's sac in the left anterior kidney is observed. C) Section of a control wild type fry. Arrows indicate glomeruli and arrowheads indicate tubules. The figure which shows the fry which transfected Pkd2 (DELTA) C-EGFP transiently. A) Appearance. Acknowledging trunk flexion. B) A section of the fry two days after hatching. Dilation of Bowman's sac (*) and tubule dilatation (†) in the left anterior kidney are observed. ‡ indicates an ear capsule. C) The transgenic fry enters the atrium from the left with the veins facing. The arrow indicates the atrium and the arrowhead indicates the ventricle. D) Control wild type. The vein enters the atrium from the right. The figure which shows the Southern blotting result of the systematic transgenic medaka. A) Genomes were extracted from medakas belonging to multiple lines with EF-1α-A-EGFP-Pkd1ΔN and compared. The 6.6 kb band detected by the probe after restriction enzyme treatment with EchoRI / BglII is the transgene. 1; no.1 line, 2; no.11 line, 3; plasmid EF1-α-A-EGFP-Pkd1ΔN 10 pg, 4; 100 pg, 5; 1000 pg. B) Genomes were extracted from medaka belonging to multiple strains with TRE2-EGFP-Pkd1ΔN and compared. The 6.6 kb band detected by the probe after restriction enzyme treatment with EchoRI / BglII is the transgene. 1; no. 7 strain, 2; no. 10 strain, 3; no. 15 strain, 4; plasmid TRE2-EGFP-Pkd1ΔN 10 pg, 5; 100 pg, 6; 1000 pg. C) Genomes were extracted from medakas belonging to multiple lines with EF-1α-A-Pkd2ΔC-EGFP and compared. A 2.3 kb band detected by the probe after restriction enzyme treatment with BamHI is the transgene. 1; no.4 strain, 2; no.7 strain, 3; no.11 strain, 4; no.12 strain, 5; no.18 strain, 6; plasmid EF-1α-A-Pkd2ΔC-EGFP 10pg, 7 ; 100 pg, 8; 1000 pg. D) Genomes were extracted from medaka belonging to multiple strains with TRE2-Pkd2ΔC-EGFP and compared. A 2.3 kb band detected by the probe after restriction enzyme treatment with BamHI is the transgene. 1; no. 17 strain, 2; no. 24 strain, 3; plasmid TRE2-Pkd2ΔC-EGFP 10pg, 4; 100pg, 5; 1000pg. The figure which shows RT-PCR result of the systematic transgenic medaka. A) EF-1α-A-EGFP-Pkd1ΔN no.1 strain. L; 1 kb plus DNA ladder (Invitrogen), 1; liver cDNA, 2; kidney cDNA, 3; liver RNA, 4; kidney RNAB) EF-1α-A-Pkd2ΔC-EGFP no.12 strain. L; 1kb plus DNA ladder (Invitrogen), 1; liver cDNA, 2; kidney cDNA, 3; liver RNA, 4; kidney RNA The figure which shows the western blotting result of the systematic transgenic medaka. Proteins were extracted from medaka livers belonging to the EF-1α-A-rtTA and EF-1α-A-tTA strains, and their expressions were compared. 1; EF-1α-A-tTA no.6 strain Protein 0.15μl, 2; 1.5μl, 3; 0.5μl, 4; EF-1α-A-tTA no.3 strain 0.15μl, 5; 1.5 μl, 6; 0.5 μl, L; MagicMark XP Western Protein Standard (Invitrogen), 7; EF1-α-A-rtTA strain 0.15 μl, 8; 1.5 μl, 9; 0.5 μl, 10; EF1-α- A-tTA no.18 strain 0.15 μl, 11; 1.5 μl, 12; 0.5 μl. The arrow (→) indicates the 37 kDa target protein. The figure which shows the tissue slice of the adult fish of EF-1 (alpha) -A-Pkd2 (DELTA) C-EGFP no.12 strain | stump | stock. HE-stained section of a 7-month heterozygous adult fish. A) Weak expansion (40x). A renal cyst is found in the right kidney. * Indicates a ureter (corresponding to a human ureter). B) Continuation of serial sections. Multiple renal cysts are also found in the left kidney. C) Strong magnification (100x). D) Control wild type (40x). The figure which shows the tissue section | slice of Tet-On Pkd2 (DELTA) C-EGFP adult fish. This is a HE-stained section of an adult fish 4 months after a genotype (rtTA /-, Pkd2ΔC /-) in which both transcription factors and response genes are heterogeneous. A) Weak expansion (40x). Cystic cystic changes are observed in the right kidney. * Indicates a ureter (corresponding to a human ureter). B) Continuation of serial sections. The right renal cyst fuses into a large cyst, but there is no obvious cyst in the left kidney. C) Strong magnification (100x).

Claims (8)

A polycystic kidney model small fish,
The small fish is selected from the group consisting of zebrafish, medaka, goldfish, loach, tiger puffer and rainbow trout,
A model small fish, which retains an exogenous mutant gene having a deletion mutation that causes a deletion on the C-terminal side of a protein encoded by a PKD2 gene derived from a fish of the same species as the small fish so that a dominant negative effect can be expressed. A medaka, a model small fish according to claim 1. The model small fish according to claim 2, wherein the deletion mutation is a mutation that deletes at least a part of exons 12 to 15 of the medaka PKD2 gene. A screening method for external factors associated with the formation or development of cystic kidneys , comprising:
A step of providing an external factor to be screened to the polycystic kidney model small fish according to any one of claims 1 to 3 ,
Evaluating the promoting or inhibiting action on the formation and / or progression of renal cysts in the medaka to which the external factor has been applied;
A screening method comprising:   The screening method according to claim 4, wherein the external factor is a compound or an environmental factor other than the compound. The screening method according to claim 4 or 5 , wherein the evaluation step is a step of evaluating an action of suppressing formation or progress of the renal cyst . The screening method according to any one of claims 4 to 6, wherein the external factor imparting step is a step of imparting the external factor during the development of the polycystic kidney model small fish. The screening method according to any one of claims 4 to 7, wherein the external factor imparting step is a step of supplying a gas containing the external factor to the polycystic kidney model small fish.