Knock-in non-human animal, method for evaluating reproductive function using same, and method for screening factors causing reproductive toxicity

Abstract

Disclosed are a non-human animal into which a reporter gene under the control of an acrosine promoter has been knocked in, a method for evaluating reproductive function using the non-human animal, and a method for screening factors causing reproductive toxicity.

Description

Knock-in non-human animal, method for evaluating reproductive function using the same, and method for screening reproductive toxicity factors

[0001] The present invention relates to a knock-in non-human animal, a method for evaluating reproductive function using the same, and a method for screening reproductive toxicity factors.

[0002] Reproductive toxicity refers to a phenomenon in which exogenous chemical substances, physicochemical factors, or endogenous physiological factors alter the reproductive capacity of parents or their born offspring. Changes in reproductive capacity include morphological and functional abnormalities, as well as damage to embryos and fetuses.

[0003] In conventional one-generation reproductive toxicity tests, it has been common practice to confirm fertility (reproductive toxicity) by administering, for example, a chemical substance to parental male and female experimental animals and allowing them to mate, and then observe what influence the substance has on the development of the resulting offspring up to sexual maturation. If necessary, gonads were excised after euthanasia and subjected to histopathological examination. Patent Document 1 discloses an Mvh-lacZ transgenic mouse having a spermatogenesis inhibitory effect and used for monitoring reproductive toxic chemicals. However, since insertion patterns and copy numbers of the genome can vary in transgenic mice, the present inventors have attempted to generate knock-in mice for observing reproductive toxicity (Non-Patent Documents 1 and 2).

[0004] Japanese Unexamined Patent Application Publication No. 2001-8577

[0005] Fukunaga Hisanori, "Spermatogenesis after environmental radiation exposure and effects on the next generation", 2022 Annual Report of Emergent Research Support Project; Fukunaga Hisanori, "Precise spatial control by microbeam and preservation of spermatogenic ability - Development of microbeam radiation therapy", Nakayama Foundation for Human Science, 2022 (Reiwa 4) Nakayama Prize Encouragement Award "Human Science of Spatial Design"

[0006] Incidentally, conventional reproductive toxicity tests, which require mating and euthanasia of experimental animals, involve uncertainties regarding mating compatibility and pregnancy, necessitating a large number of experimental animals and associated costs for their care and management. Furthermore, observing and evaluating how toxicity occurs in the gonads requires pathological and histological examination after euthanasia, making it difficult to regularly or over the long term monitor the process of reproductive function loss and recovery in the same individual in real time.

[0007] Therefore, this disclosure aims to provide a knock-in non-human animal that enables long-term evaluation of the reproductive function of the same individual, as well as a method for evaluating reproductive function using the same and a method for screening factors of reproductive toxicity.

[0008] This disclosure relates, for example, to the following [1] to

[12] : [1] A method for screening factors for reproductive toxicity, comprising exposing a non-human animal in which a reporter gene under the control of the acrosin promoter has been knocked in to a substance or environment suspected of causing reproductive toxicity, and measuring the expression level of the reporter gene in the testes of the non-human animal exposed to the substance or environment. [2] The screening method according to [1], wherein the reporter gene is a luciferase gene. [3] The screening method according to [1] or [2], wherein the non-human animal is a non-human animal in which the reporter gene has been knocked in in a homozygous manner. [4] The screening method according to any one of [1] to [3], wherein the non-human animal is a non-human mammal. [5] The screening method according to any one of [1] to [4], wherein the non-human animal is a rodent. [6] A non-human animal in which a reporter gene under the control of the acrosin promoter has been knocked in. [7] The non-human animal according to [6], in which the reporter gene has been knocked in in a homozygous manner. [8] A method for evaluating reproductive function, comprising measuring the expression level of the reporter gene in the testes of a non-human animal as described in [6] or [7]. [9] Mouse spermatogenic cells in which a reporter gene under the control of the acrosin promoter is knocked into the ROSA26 locus.

[10] A method for screening factors for reproductive toxicity, comprising exposing spermatogenic cells collected from a non-human animal as described in [6] or [7] to a substance or environment suspected of causing reproductive toxicity, and measuring the expression level of the reporter gene in the cells exposed to the substance or environment.

[11] The screening method according to

[10] , wherein the reporter gene is a luciferase gene.

[12] A vector for both heterozygous and homozygous expression, comprising an acrosin promoter and a reporter gene.

[0009] This disclosure makes it possible to stably and quantitatively evaluate the reproductive function of the same individual over a long period of time. This disclosure allows for the long-term and quantitative measurement of reproductive toxicity caused by exogenous chemicals or various physicochemical factors in the same individual. Furthermore, it enables real-time evaluation of the degree of differentiation and maturation of spermatogenic cells without euthanasia, thereby reducing the number of animals used and the costs of animal husbandry for reproductive toxicity evaluation.

[0010] Furthermore, when manipulating genes related to reproductive function in experimental animals, there is generally a significant risk of creating models that impair normal reproductive function. In particular, when genes related to reproductive function are modified in a homozygous manner, the risk of experimental animals losing normal reproductive function becomes even more serious. These risks were also a concern in the attempts to create knock-in mice described in Non-Patent Documents 1 and 2. However, according to this disclosure, it was found that even when the reporter gene inserted downstream of the acrosin promoter is knocked in a homozygous manner, as well as a heterozygous manner, the resulting knock-in models possess normal reproductive function. Therefore, the vector according to this disclosure makes it possible to create both heterozygous and homozygous model animals with normal reproductive function. It was found that using these model animals allows for broad screening of genotoxicity related to reproductive function, including loss of heterozygosity, depending on exposure conditions to various test substances and physicochemical factors.

[0011] Figure 1 is a vector map of a vector (a vector having the nucleotide sequence represented by Sequence ID No. 1) for knocking in the luciferase gene under the control of the acrosin promoter. Figure 2 shows the long-term luminescence imaging results of genetically modified mice (ROSA26KI(Acr-Luc) / +). Figure 3 is a graph showing the relative total photon flux over time in the testes of genetically modified mice (ROSA26KI(Acr-Luc) / + and ROSA26KI(Acr-Luc) / (Acr-Luc)). Figure 4(A) is a graph showing the relative total photon flux over time in the testes of genetically modified mice (ROSA26KI(Acr-Luc) / +) that have not been irradiated with X-rays. Figure 4(B) is a graph of the relative total photon flux over time in the testes of genetically modified mice (ROSA26KI(Acr-Luc) / +) irradiated with X-rays. Figure 5(A) is a graph of the relative total photon flux over time in the testes of genetically modified mice (ROSA26KI(Acr-Luc) / +) that were not administered TSA. Figure 5(B) is a graph of the relative total photon flux over time in the testes of genetically modified mice (ROSA26KI(Acr-Luc) / +) that were administered TSA. Figure 6 shows the results of three-dimensional emission intensity imaging in the testes of genetically modified mice (ROSA26KI(Acr-Luc) / +). Figure 7(A) is a graph showing the results of three-dimensional luminescence intensity imaging in the testes of genetically modified mice (ROSA26KI(Acr-Luc) / +) that were not administered TSA. Figure 7(B) is a graph showing the results of three-dimensional luminescence intensity imaging in the testes of genetically modified mice (ROSA26KI(Acr-Luc) / +) that were administered TSA. Figure 8 is a graph showing the relationship between the number of spermatogenic cells and the amount of luciferase emission collected from genetically modified mice (ROSA26KI(Acr-Luc) / +). Figure 9 is a graph showing the exponential decay of cell viability in a spermatogenic cell suspension collected from genetically modified mice (ROSA26KI(Acr-Luc) / +). Figure 10 is a graph showing the change in cell viability over time in a spermatogenic cell suspension to which bisphenol A (BPA) was added.Figure 11 is a graph showing the cell viability at each measurement time in a spermatogenic cell suspension to which bisphenol A (BPA) has been added.

[0012] The following describes the forms for implementing this disclosure, but this disclosure should not be construed as being limited to the following embodiments.

[0013] <Knock-in Non-Human Animals> One embodiment of the present disclosure relates to a non-human animal (hereinafter also simply referred to as "knock-in non-human animal") in which a reporter gene under the control of the acrosin promoter has been knocked in.

[0014] The statement "the reporter gene is under the control of the acrosin promoter" means that the acrosin promoter plays a role in regulating the expression of the reporter gene. Specifically, the reporter gene is inserted downstream of the acrosin promoter, and when the acrosin promoter is activated, the transcription of the reporter gene is promoted, resulting in the production of a reporter protein. In this way, the activity state of the acrosin promoter can be observed or measured through the expression of the reporter gene. Since the acrosin promoter is activated specifically in spermatogenic cells after meiosis, the reporter gene under the control of the acrosin promoter is expressed specifically in spermatogenic cells. Therefore, using a knock-in non-human animal according to one embodiment, it is possible to quantitatively measure the presence of spermatogenic cells after meiosis. Furthermore, the knock-in non-human animal according to one embodiment can also be described as "a non-human animal in which the reporter gene is expressed specifically in spermatogenic cells."

[0015] Acrosin is a type of serine protease specifically expressed in spermatogenic cells and can be isolated from humans, pigs, mice, rats, etc. Natural acrosin is synthesized as pro-acrosin, and after processing, it becomes active and is known to degrade the zona pellucida of the egg, thereby assisting sperm in penetrating the zona pellucida. The acrosin promoter is a polynucleotide that has the activity to regulate the transcription of the acrosin gene, and may be a polynucleotide of about 2.4 kbp upstream of the start codon, and may be the polynucleotide represented by SEQ ID NO: 2. The acrosin promoter may be of mammalian origin, for example, of human, pig, mouse, or rat origin.

[0016] In one embodiment, the knock-in non-human animal may be a homozygous organism possessing the knock-in allele in a homozygous manner, or a heterozygous organism possessing the knock-in allele in a heterozygous manner. When a heterozygous organism is used as the knock-in non-human animal, the presence or absence of loss of heterozygosity can be investigated, for example, by measuring the expression level of the reporter gene after exposing the knock-in non-human animal to the test substance or test environment described later. More specifically, if the expression level of the reporter gene after exposure is not significantly different from the expression level of the reporter gene in a knock-in non-human animal in which loss of heterozygosity is known to have occurred, it can be considered that loss of heterozygosity has occurred.

[0017] Examples of reporter genes include the β-galactosidase gene, the luciferase gene, and the gene encoding green fluorescent protein. In the vector shown in Figure 1, the luciferase gene is used as the reporter gene, and the sequence of this gene is the nucleotide sequence represented by Sequence ID No. 3.

[0018] Examples of non-human animals include mice, rats, guinea pigs, hamsters, rabbits, goats, cattle, horses, pigs, dogs, cats, and monkeys. Non-human animals may be mammals other than humans, and may be rodents. Mice are preferred as rodents, and mice such as C57 / BL / 6, ICR, and BALB / c can be used.

[0019] Knock-in non-human animals can be produced by introducing a vector containing an acrosin promoter and a reporter gene into a non-human animal. The method for introducing the vector into a non-human animal, i.e., the method for producing a knock-in non-human animal, may include introducing the knock-in vector into a host cell. To introduce the knock-in vector into a host cell, the knock-in vector may be microinjected into the nucleus of a fertilized egg, or electroporation, lipofection, or viral infection may be performed on pluripotent stem cells such as ES cells. A chimeric animal can be obtained by injecting the stem cells into which the knock-in vector has been introduced into an early embryo by a conventional method and transplanting the resulting early embryo into a surrogate parent. The obtained chimeric animal can be propagated, and from the progeny, a knock-in non-human animal in which the reporter gene has been knocked into the target allele can be obtained. More specifically, knock-in non-human animals can be produced by the method described in the examples, for example.

[0020] Examples of vectors include plasmid vectors, bacterial artificial chromosomes, yeast artificial chromosomes, retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, and piggyBac transposon vectors.

[0021] A vector for producing a knock-in non-human animal may include a 3' homologous arm and a 5' homologous arm, and may also include, for example, sequences containing exons and introns, a 3' untranslated region such as a bGH polyA addition signal, a recombinant enzyme substrate sequence, and a drug selection marker. The vector for producing a knock-in non-human animal may be the vector shown in Figure 1. The vector for producing a knock-in non-human animal may be a heterozygous expression vector or a homozygous expression vector, or a vector for both heterozygous and homozygous expression.

[0022] The 3' homologous arm is a nucleotide sequence homologous to a portion of the nucleotide sequence located downstream of the target region, and the 5' homologous arm is a nucleotide sequence homologous to a portion of the nucleotide sequence located upstream of the target region. The target region may be, for example, the mouse ROSA26 gene locus. In the vector shown in Figure 1, the 3' homologous arm is the nucleotide sequence represented by Sequence ID No. 4. In the vector shown in Figure 1, the 5' homologous arm is the nucleotide sequence represented by Sequence ID No. 5.

[0023] In Figure 1, sequences containing exon 1 and splicing acceptors correspond to sequences containing exons and introns, but are not limited to these.

[0024] Examples of recombinant enzymes include Cre, Dre, and Flp. Typically, the LoxP sequence is used for Cre, the Rox sequence for Dre, and the FRT sequence for Flp as recombinant enzyme substrate sequences.

[0025] Examples of drug selection markers include the neomycin resistance gene (neo), the hygromycin B phosphotransferase gene, the herpesvirus thymidine kinase gene (HSV-tk), and the diphtheria toxin A gene.

[0026] <Method for evaluating reproductive function> One embodiment of the present disclosure relates to a method for evaluating reproductive function, which includes measuring the expression level of a reporter gene in the testes of a knock-in non-human animal.

[0027] One embodiment of the evaluation method may include comparing the expression level of a reporter gene (reference value) in a non-human animal whose expression level for each week or month is known, or whose spermatogenic cell development level for each week or month is known, with the expression level of a reporter gene (measured value) in the testes of a knock-in non-human animal. One embodiment of the evaluation method may include determining that the reproductive function of the knock-in non-human animal used for measurement is normal if the measured value is at the same level as the reference value. For example, the measured value being at the same level as the reference value may mean that there is no significant difference between the measured value and the reference value. The reference value and the measured value may be compared for a specific week or month, or the reference value and the measured value may be compared over multiple weeks or months.

[0028] The method for measuring the expression level of a reporter gene is appropriately selected depending on the protein encoded by the reporter gene. For example, if the reporter gene encodes luciferase, luciferin can be administered to a knock-in non-human animal, and the amount of luminescence can be measured using a commercially available detector to determine the expression level of the reporter gene. Even when other reporter genes are used, the amount of the reporter gene product can be measured using known methods.

[0029] <Screening Method for Factors of Reproductive Toxicity Using Knock-in Non-Human Animals> One embodiment of the present disclosure relates to a screening method for factors of reproductive toxicity, comprising exposing a knock-in non-human animal to a substance or environment suspected of causing reproductive toxicity, and measuring the expression level of a reporter gene in the testes of the knock-in non-human animal exposed to the substance or environment (hereinafter also referred to as the "target expression level"). The screening method for factors of reproductive toxicity according to one embodiment may include a screening method for a substance suspected of causing reproductive toxicity (hereinafter also referred to as the "test substance"), and may also include a screening method for an environment suspected of causing reproductive toxicity (hereinafter also referred to as the "test environment").

[0030] The expression level of the reporter gene can be measured by the method described in <Method for Evaluating Reproductive Function>. One embodiment of the screening method may include comparing the target expression level with the expression level of the reporter gene in the testes of a non-human animal that specifically expresses the reporter gene in spermatogenic cells and has not been exposed to the test substance or the test environment (hereinafter also referred to as the "reference expression level"). One embodiment of the screening method may include comparing the target expression level and the reference expression level, and may include selecting a test substance or test environment that maintains the target expression level at the same level as the reference expression level, or increases or decreases it. If multiple target expression levels and reference expression levels are obtained, the average value of the multiple target expression levels may be compared with the average value of the multiple reference expression levels. The individual used to measure the target expression level and the individual used to measure the reference expression level may be the same or different. The target expression level and the reference expression level may be values ​​measured at a single time point, or they may be the average value of multiple expression level data measured over a specific period.

[0031] When the target expression level or its average value is 100%, 110%, or 120% or higher, with the baseline expression level or its average value set at 100%, the exposed test substance or test environment can be considered a factor that maintains, improves, or restores reproductive function. One embodiment of the screening method may include selecting a test substance or test environment that maintains, improves, or restores reproductive function.

[0032] When the target expression level or its average value is 100% or less, 90% or less, or 80% or less, with the standard expression level or its average value set at 100%, the exposed test substance or test environment can be considered a factor in reproductive toxicity. A screening method according to one embodiment may include selecting a test substance or test environment that is considered a factor in reproductive toxicity.

[0033] Non-human animals that specifically express the reporter gene in spermatogenic cells, regardless of whether or not they have been exposed to the test substance or the test environment, may be non-human animals known to have no reproductive abnormalities, or they may be non-human animals known to have reproductive abnormalities.

[0034] The test substances to be screened may be any chemical substance, and may be either naturally occurring or artificially synthesized. The test substances may be novel or known. The test substances may be chemical substances included in the hazard classifications 1 and 2 of reproductive toxic substances in the JIS classification. Examples of test substances to be screened include plastics, paints, synthetic detergents, insecticides, pharmaceuticals and their candidate compounds, pesticides, foods, food additives, nucleic acids, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, etc. Examples of test substances include specific chemical substances and their preparations, hydrogen cyanide, aniline, benzene, metallic elements (lead, mercury, chromium, arsenic, cadmium, selenium, boron, etc.), halogen elements (fluorine, chlorine, etc.), hydrogen halides (hydrogen fluoride, hydrogen chloride, etc.), radioactive elements and their compounds, hazardous substances such as yellow phosphorus, hydrofluoric acid, hydrochloric acid, nitric acid, sulfuric acid, sulfurous acid, carbolic acid, caustic alkalis, carbon monoxide, carbon dioxide, and similar hazardous substances, as well as gases, vapors, and dusts of these hazardous substances.

[0035] In non-human animals, the test substance may be administered by means of injection, such as tail vein administration or intraperitoneal administration; orally in powder or liquid form; or transdermally in cream or liquid form. The dosage may be appropriately adjusted depending on the route of administration and the test substance being administered.

[0036] The test environment may be, for example, an environment in which the subject is exposed to the test substance described above, or an environment in which stress is imposed on the knock-in non-human animal. Examples of stress include exposure to ionizing radiation such as X-rays, exposure to non-ionizing radiation such as visible light, infrared rays, and radio waves, abnormal temperatures such as heat or cold, abnormal humidity, abnormal atmospheric pressure, abnormal illumination lacking a periodic light-dark cycle, vibration, noise, weightlessness such as in outer space, infection by pathogens such as viruses and bacteria, and dieting such as starvation. The method of exposing the knock-in non-human animal to the test environment may be appropriately selected for each test environment.

[0037] <Spermatogenic Cells> One embodiment of the present disclosure relates to mouse spermatogenic cells in which a reporter gene under the control of the acrosin promoter is knocked in at the ROSA26 locus. The definitions and explanations of the acrosin promoter, reporter gene, and "the reporter gene is under the control of the acrosin promoter" are as described in the section <Knock-in Non-Human Animals>.

[0038] The spermatogenic cells described above can be collected from the testes of mice in which a reporter gene under the control of the acrosin promoter has been knocked into the ROSA26 locus. Known methods may be used for collection. For example, one method involves finely cutting the seminiferous tubules in the testes and extracting the spermatogenic cells, which can be specifically carried out as described in Example 5.

[0039] <Screening Method for Factors of Reproductive Toxicity Using Spermatogenic Cells Derived from Knock-in Non-Human Animals> One embodiment of the present disclosure relates to a screening method for factors of reproductive toxicity, comprising exposing spermatogenic cells collected from a knock-in non-human animal to a substance suspected of being reproductively toxic (test substance) or an environment (test environment), and measuring the expression level of a reporter gene in the cells exposed to the substance or environment (hereinafter also referred to as "target cell expression level"). The number of viable spermatogenic cells correlates with the target cell expression level. Therefore, by measuring the target cell expression level, it is possible to evaluate the effect of the test substance or test environment on the viability of spermatogenic cells, i.e., the reproductive toxicity of the test substance or test environment. Furthermore, the screening method according to this embodiment may also include exposing spermatogenic cells collected from a knock-in non-human animal to both the test substance and the test environment. By exposing spermatogenic cells to a combination of the test substance and the test environment, it becomes possible to evaluate the additive and synergistic effects of both. In this disclosure, the term "substance suspected of being reproductively toxic" is used synonymously with "substance suspected of causing reproductive toxicity."

[0040] The screening method according to this embodiment may include collecting spermatogenic cells from knock-in non-human animals. Examples of collection methods include those described above.

[0041] Examples of the test substance in the present embodiment include the aforementioned test substances. There is no particular limitation on the method for bringing spermatogenic cells into contact with a test substance, and examples thereof include a method of adding the test substance to a cell suspension containing spermatogenic cells.

[0042] The test environment in the present embodiment may be an environment that applies stress to spermatogenic cells. Examples of the stress include irradiation with ionizing radiation such as X-rays, irradiation with non-ionizing radiation such as visible light, infrared rays, and radio waves (e.g., radio waves), abnormal temperature, infection with pathogens such as viruses and bacteria, and depletion of nutrients in a culture solution. The method for exposing spermatogenic cells to the test environment may be appropriately selected depending on the test environment.

[0043] The method for measuring the expression level in target cells is appropriately selected depending on the protein encoded by the reporter gene. For example, when the reporter gene encodes luciferase, after adding a reagent containing a luciferin substrate to the cells, the amount of luminescence can be measured using a commercially available detector (e.g., a luminescence microplate reader), and the measured value can be used as the expression level of the reporter gene. Even when other reporter genes are used, the amount of the reporter gene product can be measured by a known method.

[0044] The screening method according to the present embodiment may include comparing the expression level in target cells with the expression level of the reporter gene in spermatogenic cells collected from a knock-in non-human animal that has not been exposed to any test substance or test environment (hereinafter also referred to as "reference cell expression level"). The screening method may include comparing the expression level in target cells and the reference cell expression level, and may include selecting a test substance or test environment that maintains the expression level in target cells at the same level as the reference cell expression level, or increases or decreases the expression level in target cells. When a plurality of expression levels in target cells and a plurality of reference cell expression levels are obtained respectively, an average value of the plurality of expression levels in target cells may be compared with an average value of the plurality of reference cell expression levels. Each of the expression level in target cells and the reference cell expression level may be a value measured at a predetermined time point after exposure to the test substance or the test environment (for example, after 2 to 24 hours have elapsed).

[0045] With the reference cell expression level or the average value thereof defined as 100%, when the target cell expression level or the average value thereof is 100% or more, 110% or more, or 120% or more, the exposed test substance or test environment can be regarded as a factor that maintains, improves or restores reproductive function. The screening method according to the present embodiment may comprise selecting a test substance or a test environment that maintains, improves or restores reproductive function.

[0046] With the reference cell expression level or the average value thereof defined as 100%, when the target cell expression level or the average value thereof is 100% or less, 90% or less, or 80% or less, the exposed test substance or test environment can be regarded as a factor causing reproductive toxicity. The screening method according to the present embodiment may comprise selecting a test substance or a test environment that has been regarded as a factor causing reproductive toxicity.

[0047] The above-mentioned target cell expression level and reference cell expression level may be the absolute value of the measured expression level of the reporter gene, or may be a relative value calculated based on the expression level of the reporter gene at the start of measurement (e.g., cell viability).

[0048] The present disclosure will be described in more detail below with reference to Examples, but the present disclosure should not be construed as being limited to the following Examples.

[0049] <Creation of Genetically Modified Mice> (1) Construction of Homologous Recombination Vector The homologous recombination vector was constructed by inserting a splicing acceptor (SA), a bovine growth hormone polyA addition signal (bGH-polyA), an acrosin promoter (Acr promoter), a luciferase gene (Luc2), and a neomycin (neo) resistance gene flanked by FRT between the 5' homologous region and the 3' homologous region (Figure 1). The sequence from nucleotide positions 6306 to 6376 of the sequence represented by Sequence ID No. 1 is exon 1 in Figure 1. The sequence from nucleotide positions 7521 to 7637 of the sequence represented by Sequence ID No. 1 is the splicing acceptor in Figure 1. The sequences from nucleotide positions 7638 to 7862 and from nucleotide positions 11881 to 12105 of the sequence represented by Sequence ID No. 1 are the bovine growth hormone polyA addition signal in Figure 1. The sequence from position 9017 to 9151 of the nucleotide sequence represented by Sequence ID No. 1 is Gm36221 ncRNA ex2 in Figure 1. The sequence from position 9859 to 10052 of the nucleotide sequence represented by Sequence ID No. 1 is Gm36221 ncRNA ex1 in Figure 1. The sequences from position 12147 to 12180 and from position 13839 to 13872 of the nucleotide sequence represented by Sequence ID No. 1 are FRT in Figure 1. (2) Establishment of drug-resistant ES cell clones To obtain drug-resistant ES cell clones, homologous recombination vectors were introduced into ES cells (C57BL / 6N-derived RENKA strain) by electroporation, and colonies of drug-resistant ES cell clones formed by drug selective culture were isolated. Next, genomic DNA was purified and extracted from the isolated ES cell clones, and homologous recombinants were selected by PCR using it as a template. For the PCR tests, PCR was performed using the forward primer set shown in SEQ ID NO: 6 and the reverse primer set shown in SEQ ID NO: 7, and the forward primer set shown in SEQ ID NO: 8 and the reverse primer set shown in SEQ ID NO: 9. Furthermore, homologous recombinants selected by PCR were analyzed by Southern blotting with a Neo probe to obtain homologous recombinant ES cell clones. (3) Chimeric mouse production by aggregation method Chimeric embryos were produced by aggregation using homologous recombinant ES cell clones and 8-cell stage embryos of the ICR line.Pregnant mice that had not given birth were cesarean sectioned after their expected delivery date to the recipient female mice into which these chimeric embryos had been transplanted. The resulting chimeric mice were reared until weaning, and the chimeric rate was determined by coat color at weaning. (4) Identification of germline chimeric mice (production of F1 heterozygous mice) First-generation (F1) mice were produced by natural mating of chimeric mice with wild-type mice. Genomic DNA was collected from the body tissue of the offspring, and germline chimeric mice were identified by PCR analysis. For PCR, the forward primer set shown in SEQ ID NO: 10 and the reverse primer set shown in SEQ ID NO: 11 were used. (5) Production of drug-resistant gene-removed mice Second-generation (F2) mice were produced by mating F1 mice with CAG-Flp mice (genotype; ROSA26KI(CAG-Flp) / KI(CAG-Flp)). Genomic DNA was collected from the body tissue of the offspring, and the genotype was determined by PCR analysis to select mice (ROSA26KI(Acr-Luc) / KI(CAG-Flp)) from which the neomycin (neo) resistance gene had been removed. For PCR, the forward primer set shown in SEQ ID NO: 12 and the reverse primer set shown in SEQ ID NO: 13 were used. Next, third-generation (F3) mice were produced by naturally mating F2 heterozygous mice (ROSA26KI(Acr-Luc) / KI(CAG-Flp)) of ROSA26 locus Acr-Luc gene knock-in mice with wild-type mice. Genomic DNA was collected from the body tissue of the offspring, and the genotype was determined by PCR analysis to obtain mice (ROSA26KI(Acr-Luc) / +) that did not have the ROSA26KI(CAG-Flp) allele. In PCR, the forward primer set shown in SEQ ID NO: 14 and the reverse primer set shown in SEQ ID NO: 15 were used. By crossing ROSA26KI(Acr-Luc) / + with other ROSA26KI(Acr-Luc) / (Acr-Luc) strains, homozygotes (ROSA26KI(Acr-Luc) / (Acr-Luc)) were obtained.

[0050] <Luminescence Imaging> (Example 1) Time-course evaluation of fertility (spermatogenesis) of the same male individual Figure 2 shows the luminescence imaging results (luminescence images) of genetically modified mice (ROSA26KI(Acr-Luc) / +) using IVIS® Spectrum CT. Luciferin was administered to the tail vein of the genetically modified mouse, and imaging was performed under isoflurane anesthesia. By measuring the same individual over time, the physiological process of sexual maturation could be evaluated simply and quantitatively. Figure 3 shows the results of analyzing the obtained luminescence images. In the obtained luminescence images, a circular ROI was placed so that the entire testis was included, and the total photon flux passing through the ROI was measured. For the total photon flux obtained for each week of age, the relative value (relative total photon flux) was calculated with the total photon flux at the start of measurement (week 0) set to 1, and the graph in Figure 3 was created. Controls 1 to 3 in Figure 3 correspond to controls 1 to 3 in Figure 2, respectively, and all are data from heterozygous mice. On the other hand, Homo-1 in Figure 3 is data from homozygous mice. It was generally expected that reproductive function would be lost when genes in the reproductive system are modified, but it was found that the vector according to this disclosure does not lose reproductive function even when homozygous, and can be used to evaluate the fertility of the same individual over time, just as with heterozygous mice.

[0051] (Example 2) Monitoring of reproductive toxicity and recovery process with X-rays Fertility was evaluated over time in the same manner as in Example 1, except that 5 Gy (2.5 Gy x 2) of X-rays was irradiated to the lower body at the start of measurement (Week 0) and only heterozygous mice were used. The results are shown in Figure 4(B). For reference, the data for controls 1 to 3 of Example 1 are shown in Figure 4(A). As shown in Figure 4(B), although luminescence was observed in the testes one or two weeks after X-ray irradiation, the luminescence disappeared or decreased around four to five weeks. Subsequently, recovery of luminescence was observed from around week 6. In this way, the reproductive toxicity of X-rays could be evaluated simply and quantitatively from the luminescence intensity and distribution, which were proportional to the number of spermatogenic cells in the same individual.

[0052] (Example 3) Analysis of reproductive toxicity with the histone deacetylase inhibitor trichostatin A (TSA) Data for the TSA-administered group shown in Figure 5(B) were obtained in the same manner as in Examples 1 and 2, except that TSA (150 ng / kg) was administered via tail vein one hour before the start of measurement, and only heterozygous mice were used. For reference, data for the TSA-free group obtained in the same manner as in Examples 1 and 2, except that only heterozygous mice were used, are shown in Figure 5(A). As shown in Figure 5(B), the tendency for increased luminescence intensity in the control (non-X-ray irradiated) group observed in Figure 5(A) was suppressed for more than 8 weeks after administration, suggesting that the reproductive toxicity of TSA may be long-lasting. Also, as shown in Figure 5(B), the enhancement of luminescence intensity after X-ray irradiation in the X-ray irradiated group observed in Figure 5(A) was suppressed, suggesting that inhibition of histone deacetylase may affect changes in gene expression associated with radiation exposure.

[0053] (Example 4) Three-dimensional emission intensity imaging 15 × 15 × 15 [mm] ３Except for positioning the VOI of the cubic sphere to include the entire testis (Figure 6), measuring the photon flux within the cubic sphere, and measuring the photon flux at the start of measurement (before X-ray irradiation) and 8 weeks after the start of measurement, the procedure was the same as in Example 3 to obtain data for the TSA-free group and the TSA-administered group shown in Figures 7(A) and (B), respectively. As shown in Figure 7(A), an increase in emission intensity was observed 8 weeks after the start of measurement in the control groups 1 to 3, which received neither X-ray irradiation nor TSA administration. This suggests that testicular development over time proceeded normally. As shown in Figure 7(A), a decrease in emission intensity was observed 8 weeks after the start of measurement in the 5Gy-1 and 5Gy-2 groups, which received X-ray irradiation without TSA administration (p = 0.227, paired t-test). This suggests that radiation tends to hinder testicular development. As shown in Figure 7(B), in controls 1 and 2, who were administered TSA without X-ray irradiation, the rate of increase in emission intensity was suppressed compared to controls 1 to 3 in Figure 7(A). This suggests that TSA administration has a negative effect on testicular development. As shown in Figure 7(B), in 5Gy-1 and 2, who were subjected to both X-ray irradiation and TSA administration, the rate of decrease in emission intensity was more pronounced than in 5Gy-1 and 2 in Figure 7(A), and a significant difference was observed in photon flux before X-ray irradiation and 8 weeks after irradiation (p = 0.018, paired t-test). This suggests that TSA administration may enhance the negative effect of X-ray irradiation on testicular development.

[0054] <In vitro experiment using spermatogenic cells derived from genetically modified mice> (Example 5) Preparation of cell suspension containing spermatogenic cells All of the following operations were performed on a coolant that had been chilled in a refrigerator. The testes were removed from genetically modified mice (ROSA26KI(Acr-Luc) / +) under isoflurane anesthesia and temporarily stored in RBC Lysis Buffer (pluriSelect Life Science UG&Co.KG). The tunica albuginea of ​​the testis was removed on a petri dish containing RBC Lysis Buffer, and the seminiferous tubules were transferred to another petri dish containing GL-PBS (PBS(+) (pH 7.4) containing 0.1 mg / mL polyvinyl alcohol, 5.6 mM glucose, 5.4 mM sodium lactate, and 5 mg / mL BSA). Five or six cuts were made in the seminiferous tubules, and spermatogenic cells were gently pipetted to release them. The spermatogenic cell suspension was filtered through a 42 μm nylon mesh. The filtrate was centrifuged (800 rpm, 4 minutes), the supernatant was removed, and 1 mL of GL-PBS was added and pipetted. After repeating this washing procedure a total of three times, the cells were suspended in 10 mL of GL-PBS to obtain a cell suspension containing spermatogenic cells.

[0055] (Example 6) Correlation analysis between spermatogenic cell count and luciferase luminescence The cell suspension obtained in Example 5 was serially diluted, and samples with multiple cell counts were prepared in 96-well plates. Luciferin substrate was added to each sample, and luciferase luminescence was measured using a luminescent microplate reader. As shown in Figure 8, an extremely high positive correlation (r = 0.9997, p < 0.0001) was observed between spermatogenic cell count and luminescence. This result indicates that in this measurement system, luciferase luminescence accurately reflects the spermatogenic cell count.

[0056] (Example 7) Evaluation of reproductive toxicity of bisphenol A (BPA) The cell suspension obtained in Example 5 was dispensed into 96-well plates in 198 μL portions. 2 μL of BPA solution was added to each test group to a final concentration of 100 μM, while the same amount of DMSO (a solvent) was added to the control group. The plates were left standing in a refrigerator (4°C), and luciferin substrate was added at 0, 2, 4, 8, 12, 18, and 24 hours after addition. The amount of luciferase luminescence was measured using a luminescent microplate reader. Cell viability was calculated by dividing the amount of luminescence at each time point by the amount of luminescence at 0 hours after addition. First, the results of the analysis of cell viability in the control group are shown in Figure 9. The viability of spermatogenic cells in the cell suspension decreased exponentially with a half-life of 14.4 hours. Next, the results of measuring cell viability in spermatogenic cells to which BPA was added are shown in Figures 10 and 11. As shown in Figure 10, the BPA-added group showed a accelerated decline in cell viability over time compared to the control group. Furthermore, as shown in Figure 11, the BPA-added group showed a statistically significant decrease in cell viability compared to the control group starting 4 hours after addition (p < 0.05), and its toxic effect was enhanced after 8 hours (p < 0.01). These results demonstrate that the reproductive toxicity of a test substance can be evaluated in vitro using spermatogenic cells derived from the genetically modified mice described herein.

Claims

1. A method for screening factors of reproductive toxicity, comprising exposing a non-human animal in which a reporter gene under the control of the acrosin promoter has been knocked in to a substance or environment suspected of causing reproductive toxicity, and measuring the expression level of the reporter gene in the testes of the non-human animal exposed to the substance or environment.

2. The screening method according to claim 1, wherein the reporter gene is a luciferase gene.

3. The screening method according to claim 1 or 2, wherein the non-human animal is a non-human animal in which the reporter gene has been homozygously knocked in.

4. The screening method according to claim 1 or 2, wherein the non-human animal is a non-human mammal.

5. The screening method according to claim 1 or 2, wherein the non-human animal is a rodent.

6. Non-human animals in which a reporter gene under the control of the acrosin promoter has been knocked in.

7. The non-human animal according to claim 6, wherein the reporter gene is knocked in in a homozygous manner.

8. A method for evaluating reproductive function, comprising measuring the expression level of the reporter gene in the testes of a non-human animal according to claim 6 or 7.

9. Mouse spermatogenic cells in which a reporter gene under the control of the acrosin promoter has been knocked into the ROSA26 locus.

10. A method for screening factors for reproductive toxicity, comprising exposing spermatogenic cells collected from a non-human animal according to claim 6 or 7 to a substance or environment suspected of being reproductively toxic, and measuring the expression level of the reporter gene in the cells exposed to the substance or environment.

11. The screening method according to claim 10, wherein the reporter gene is a luciferase gene.

Images