Signal transduction analysis methods for X-linked dominant diseases

Abstract

To provide a method that enables analyzing whether a signal transmission from an allele protein to a factor (preferably, the protein of a gene existing in the downstream) existing in a downstream of the allele protein in the signal transmission is performed only within a cell where the allele protein expresses, or performed via the outside of the cell where the allele protein expresses.SOLUTION: Let two X chromosomes of a nonhuman mammal be X1 and X2, a normal allele and a fluorescent protein gene exist in the X1, and a variant allele exists in the X2, a method includes: identifiably dyeing a downstream factor in a cell of the nonhuman mammal in which the variant allele is a gene indicative of a dominant inheritance character; and detecting a fluorescent protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for analyzing signal transduction pathways in vivo. The contents of all documents mentioned in this specification are incorporated herein by reference. [Background technology]

[0002] DNA methylation (more specifically, cytosine methylation) is deeply involved in various biological processes.

[0003] For example, methylation of promoter regions (especially CpG islands) plays a regulatory role in suppressing gene expression by preventing transcription factors from binding to promoter regions, and this regulatory function is considered to be one of the major control mechanisms of epigenetics. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] J Biol Chem 274:2315-2321 (1999) [Non-patent document 2] J Clin Invest 107:1093-1102 (2001) [Non-patent document 3] Development 115, 703-715 (1992) [Non-patent document 4] Endocrinology 2019;160:1348-1358 Summary of the Invention [Problem to be solved by the invention]

[0005] It is well known that mammalian sex determination involves inheriting an X chromosome from the father, resulting in females when both sex chromosomes are X chromosomes, and one of the inherited X chromosomes is inactivated. DNA methylation is involved in this X chromosome inactivation. More specifically, X chromosome inactivation occurs when almost the entire X chromosome (with the exception of the pseudoautosomal region) adopts a heterochromatin structure, and this heterochromatin structure is achieved by DNA methylation across almost the entire X chromosome.

[0006] This inactivation is thought to occur as a form of dosage compensation. In other words, males express genes necessary for viability on their only X chromosome, while females inactivate one X chromosome to avoid excessive gene expression from the two X chromosomes. In eutherian animals such as mice and humans, which X chromosome is inactivated during female embryonic development is determined randomly for each cell. However, once inactivation occurs, the inactivation state of that X chromosome remains constant throughout life under physiological conditions. While up to 25% of the genes on the inactivated X chromosome in humans are expressed on the inactivated X chromosome, almost no genes escape inactivation on the inactivated Xi in mice.

[0007] Initially, the present inventors conducted studies with the aim of developing a method for easily detecting this X chromosome inactivation.

[0008] A search for known methods for detecting X chromosome inactivation revealed the existence of a female mouse (H253 mouse) in which the lacZ gene was knocked into one X chromosome (see Non-Patent Document 3, cited above). Histochemical staining of the tissues of the H253 mouse with X-gal revealed that in a heterozygous female (XA / Xa) with an XA (normal allele) gene on the X chromosome and an Xa (mutant allele) gene, which exhibits a dominant genetic trait, cells expressing the XA gene were lacZ-positive and blue, while cells expressing the Xa gene were lacZ-negative and white, making it possible to distinguish which allele, XA or Xa, was expressed (see Figure 7). This allows the state of X chromosome inactivation to be determined cell-by-cell.

[0009] However, when applying X-gal to tissues in H253 mice, the mice are sacrificed, the tissues are excised, and X-gal histochemical staining is performed. However, X-gal coats the tissue surface. This makes immunohistochemistry impossible to confirm target gene expression. If X-gal histochemical staining is performed after immunohistochemistry, lacZ denatures, making X-gal histochemical staining impossible. For these reasons, the H253 mice cannot be used for analysis. Furthermore, the H253 mice were developed by randomly introducing transgenes and then selecting those that happened to have the transgene introduced on the X chromosome (see Non-Patent Document 3, supra). Therefore, further improvement using similar techniques was practically impossible.

[0010] Therefore, we further investigated a novel method different from that used in H253 mice, which would enable immunostaining using tissues extracted from sacrificed mice. As a result, we discovered the possibility of creating an animal model in which X chromosome inactivation could be detected by knocking in a specific fluorescent protein gene at a specific site on the X chromosome, and continued our investigation.

[0011] In this study, the present inventors generated a female non-human mammal in which the two X chromosomes of the female non-human mammal are designated X1 and X2, with a normal allele and a fluorescent protein gene present on X1 and an allele but no fluorescent protein gene present on X2, and found that by identifiably staining a factor present downstream of the allele protein (downstream factor) and detecting the fluorescent protein, it may be possible to analyze whether signal transmission from the allele protein to the downstream factor occurs exclusively within the cell in which the allele protein is expressed, or whether signal transmission occurs via signal transmission from the cell in which the allele protein is expressed to another cell. Therefore, further studies were conducted on this analytical method. [Means for solving the problem]

[0012] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. Signal transduction from an allele protein to a factor (downstream factor) present downstream of the allele protein in signal transduction only in cells in which the allele protein is expressed, or via the extracellular route where the allele protein is expressed, A method for analyzing If the two X chromosomes of a female non-human mammal are X1 and X2, X1 contains the normal allele and the fluorescent protein gene. The mutant allele on X2 exists, the mutant allele is a gene that exhibits a dominant inherited trait; In female non-human mammalian cells, distinguishably staining the downstream factor and detecting the fluorescent protein; method. Section 2. Item 2. The method according to Item 1, wherein the downstream factor is a protein (downstream protein) of a gene that is located downstream of the allele protein in signal transduction. Section 3. The fluorescent protein gene present in X1 is the fluorescent protein gene knocked into X1, The knock-in site is an intron site of a gene on the X chromosome. Item 1 or 2. The method according to item 1 or 2. Section 4. The intron region of the gene on the X chromosome where the fluorescent protein gene is knocked in is The intron region of Moesin, The method according to item 3. Section 5. Item 5. The method according to Item 3 or 4, wherein the knock-in fluorescent protein gene is a green fluorescent protein gene, a yellow fluorescent protein gene, or a red fluorescent protein gene. Section 6. Item 6. Analyzing signal transduction from an allele protein to a factor (downstream factor) present downstream of the allele protein in signal transduction by a method according to any one of Items 1 to 5; and When it is analyzed that the signal transduction is carried out outside the cells in which the allele protein is expressed, applying a test substance to the female non-human mammal and then analyzing the signal transduction from the allele protein to a factor (downstream factor) present downstream of the allele protein in the signal transduction by the method described in any one of Items 1 to 5. A method for screening for a substance that inhibits the signal transduction, comprising: [Effects of the Invention]

[0013] The present invention provides a method for analyzing whether signal transmission from an allele protein to a factor (preferably a protein of a gene located downstream) located downstream of the allele protein in signal transduction occurs only within the cell in which the allele protein is expressed, or whether signal transmission occurs via the outside of the cell in which the allele protein is expressed. [Brief explanation of the drawings]

[0014] [Figure 1] A schematic diagram of the construct "CAG promoter-tdTomato-pA" is shown. Schematics of the target allele and knock-in allele are also shown. [Figure 2] This shows the signal transduction relationship between PHEX and FGF23, which is thought to be involved in the development of X-linked hypophosphatemic rickets (XLH). [Figure 3] Frozen sections of bone tissue from female X1tdTomato+, PHEXwt / X2tdTomato-, and PHEXwt mice (upper right and left of the figure), which have the tdTomato gene and a normal PHEX gene on the X chromosome, and female X1tdTomato+, PHEXwt / X2tdTomato-, and PHEXmut mice (lower right and left of the figure), which have the tdTomato gene and an inactive mutant PHEX gene on the X chromosome, were immunostained with an anti-FGF23 antibody and observed under a fluorescence microscope (upper right and lower right of the figure), and further stained with DAPI for nuclei (upper left and lower left of the figure). [Figure 4] The chromosomes of a heterozygous female (XA / Xa) with the XA (normal allele) gene and the Xa (mutant allele) gene on the X chromosome are shown, along with the changes in phenotype (PA, Pa) depending on whether or not intercellular signaling occurs. [Figure 5]In UMR-106 cells, a cultured osteocyte lineage cell lineage (which expresses the osteocyte-specific transcription factor Osterix along with FGF23), when Osterix expression was suppressed using siRNA against Osterix, Osterix expression was suppressed (left), and FGF23 expression subsequently decreased (right). This indicates that the FGF23 gene is located downstream of the Osterix gene. [Figure 6] The top panel shows a female mouse (X1tdTomato+ / PHEXwt / X2tdTomato- / PHEXwt mouse) with a normal phenotype. The top left panel shows immunostaining with an anti-Osterix antibody, with Osterix-expressing cells stained green. The top right panel shows additional nuclei stained with DAPI, allowing the location of the cells to be confirmed. In a female mouse (X1tdTomato+ / PHEXwt / X2tdTomato- / PHEXwt mouse) with a normal phenotype, Osterix expression was confirmed regardless of the presence or absence of tdTomato expression. This indicates that Osterix is ​​expressed in all normal bone cells. The bottom panel shows a female mouse (X1tdTomato+ / PHEXwt / X2tdTomato- / PHEXmut mouse) with a rickets phenotype. Normal cells are tdTomato-positive, while cells with the pathogenic mutant PHEX gene (pathological cells) are tdTomato-negative. Because Osterix is ​​more strongly expressed in tdTomato-negative cells than in tdTomato-positive cells, it can be concluded that the signaling pathway from the mutant PHEX gene to Osterix is ​​completed only within tdTomato-negative cells. [Figure 7] This shows the relationship between the inactivation (methylation) state of the X chromosome and the observed color in female mice (H253 mice) in which the lacZ gene was knocked into one of the X chromosomes (Non-Patent Document 3). DETAILED DESCRIPTION OF THE INVENTION

[0015] Each embodiment included in the present disclosure will be described in more detail below. The present disclosure provides a method capable of analyzing whether signal transmission from an allele protein to a factor (downstream factor) present downstream of the allele protein in signal transmission occurs only within the cell in which the allele protein is expressed, or whether it occurs via transmission from the cell in which the allele protein is expressed to another cell (intercellular transmission). However, the present disclosure is not limited thereto, and includes all that is disclosed herein and that can be recognized by a person skilled in the art.

[0016] For convenience of explanation, the female non-human mammal used in the method encompassed by the present disclosure is a female non-human mammal in which, when the two X chromosomes are designated X1 and X2, a normal allele and a fluorescent protein gene are present on X1, and a mutant allele is present on X2. This female non-human mammal may be referred to as the female non-human mammal of the present disclosure. For convenience of explanation, the normal allele may be designated as A (or XA, which also means that it is present on the X chromosome), and the mutant allele may be designated as a (or Xa, which also means that it is present on the X chromosome). According to this designation, the female non-human mammal of the present disclosure is a heterozygous female (X) with an A (normal allele) gene on the X chromosome and an a (mutant allele) gene that exhibits a dominant genetic trait. A / X a ) It can also be said that the A gene is present in X1, the a gene is present in X2, and the fluorescent protein gene is present in X1.

[0017] It is preferable that the A gene present in X1 or the a gene present in X2 is a gene that exhibits a dominant genetic trait.

[0018] The fluorescent protein gene present in X1 is preferably a fluorescent protein gene knocked into X1. The fluorescent protein is not particularly limited, but preferred examples include a green fluorescent protein gene, a yellow fluorescent protein gene, and a red fluorescent protein gene. More specific examples of fluorescent proteins include Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP, GFP, TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, HyPer, TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, and mBa Examples include nana, KusabiraOrange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed, mRasberry, mPlum, PS-CFP, Dendra2, Kaede, EosFP, KikumeGR, and td-Tomato.

[0019] The site where the fluorescent protein gene is knocked in is preferably an intron site of a gene on the X chromosome. A gene present on the X chromosome is preferably one that allows a knockout non-human mammal to survive, and more preferably one that does not exhibit any particular phenotype when knocked out. A preferred example of such a gene is the moesin (Membrane-Organizing Extension Spike protein) gene. The accession numbers for the moesin protein are NP_002435 for humans and NP_034963 for mice. When the site where the fluorescent protein gene is knocked in is an intron site of the moesin gene, knocking in into the intron between exon 2 and exon 3 is particularly preferred.

[0020] When the site where the fluorescent protein gene is knocked in is an intron site of a gene on the X chromosome, the gene may be the normal allele A, but it is preferable that this is not the case.

[0021] If a fluorescent protein gene is knocked into, for example, a region on the X chromosome where no gene is encoded, there is a risk that the fluorescent protein will not be sufficiently expressed. However, if the site where the fluorescent protein gene is knocked into is an intron region of the gene on the X chromosome, this risk is reduced, which is preferable.

[0022] The non-human mammal is not particularly limited, but is preferably, for example, a rodent, and among these, mice, rats, hamsters, etc. are particularly preferred.

[0023] By using the cells of the female non-human mammal of the present disclosure, it is possible to analyze whether signal transmission from the allele (A or a) protein to a factor (downstream factor) present downstream of the allele protein in signal transduction occurs only within the cell in which the allele protein is expressed, or whether it occurs via an extracellular pathway in which the allele protein is expressed. This analytical method is also preferably encompassed by the present disclosure. In addition, this analytical method may also be referred to as the method of the present disclosure.

[0024] The downstream factor is not particularly limited as long as it is a compound responsible for signal transduction, and examples thereof include low molecular weight compounds, peptides, proteins, and nucleic acids. Other examples include hormones, enzymes, and receptors. Although not particularly limited, the downstream factor is preferably a protein (downstream protein) of a gene that is present downstream of the allele protein in signal transduction.

[0025] The cells of the female non-human mammal of the present disclosure used in the methods of the present disclosure may be cells in the female non-human mammal of the present disclosure, or may be isolated or cultured cells isolated (and optionally cultured) from the female non-human mammal of the present disclosure.

[0026] The method of the present disclosure includes distinguishably staining the downstream factor and detecting the fluorescent protein in cells of the female non-human mammal of the present disclosure.

[0027] The downstream factor is preferably stained by immunostaining. That is, an antibody capable of specifically detecting the downstream protein is labeled, and the labeled antibody is used to stain the downstream factor in an identifiable manner. Examples of immunostaining methods include autoradiography (using an antibody labeled with a radioisotope), enzyme antibody techniques (using an antibody labeled with an enzyme used in a color reaction), and fluorescent antibody techniques (using an antibody labeled with an antibody dye).

[0028] Furthermore, the antibody used for immunostaining may be either a polyclonal antibody or a monoclonal antibody, or may be a fragment thereof (such as Fab or F(ab')2) that has specific detection ability.

[0029] The method of the present disclosure can be used to determine whether signal transduction from an allele protein to a downstream factor occurs only in cells in which the allele protein is expressed, or It is possible to analyze whether the allele protein is transmitted from the cell in which it is expressed to other cells (intercellular transmission). This point will be explained in more detail with an overview shown in Figure 4. Note that in Figure 4, the tdTomato gene is used as the fluorescent protein gene, but this is an example, and as mentioned above, the fluorescent protein gene is not particularly limited.

[0030] Figure 4 illustrates the chromosomes of a heterozygous female (XA / Xa) with the XA (normal allele) gene and the Xa (mutated allele) gene on the X chromosome. In Figure 4, Xa represents a dominant genetic trait. Cells 1A and 1B show the relationship between genotype and phenotype in each cell when intercellular signaling is not considered. In cell 1A, the XA and tdTomato genes are inactivated by methylation, and Xa is expressed, resulting in a Pa phenotype. The absence of tdTomato expression results in a white color. Cell 1B expresses the XA and tdTomato genes, but Xa is inactivated by methylation, resulting in a PA phenotype. The expression of the tdTomato gene results in a red color. Cells 2A and 2B show the absence of intercellular signaling. Because intercellular signaling is absent, white cells exhibit the Pa phenotype, as in cells 1A and 1B, while red cells exhibit the PA phenotype. Cells 3A and 3B show the presence of intercellular signaling. The white 3A cells naturally express Xa, so they exhibit a Pa phenotype. Although the red 3B cells do not express Xa, signals enter them from the white 3A cells through intercellular signaling, and because Pa is dominant over PA, they exhibit a Pa phenotype. There is also intercellular signaling from the 3B cells to the 3A cells, but because Pa is dominant over PA, the phenotype remains Pa. From the above, the results obtained using this method indicate the following: (i) If the mutant phenotype (Pa) is expressed only in tdTomato-negative cells, the signaling pathway from the Xa gene to the mutant phenotype (Pa) is transmitted only intracellularly. (ii) If the mutant phenotype (Pa) is expressed in both tdTomato-positive and -negative cells, the signaling pathway from the Xa gene to the mutant phenotype (Pa) is mediated by intercellular signaling (i.e., the signal is transmitted outside the cell), possibly via paracrine factors, although this is not intended to be limiting.

[0031] As can be seen from the example in Figure 4, the method of the present disclosure makes it possible to analyze whether a protein allele is expressed only within the cell, or whether the protein allele is expressed between cells. The explanation of Figure 4 above will be generalized as follows. (I) When the downstream factor is expressed only in fluorescent protein-negative cells, the signal pathway from the Xa gene to the downstream factor is completed only in fluorescent protein-negative cells (i.e., Xa gene-expressing cells). (II) When the downstream factor is expressed in both fluorescent protein-positive and -negative cells, the signaling pathway from the Xa gene to the downstream factor is intercellular (i.e., the signal is transmitted outside the cell) (probably, but not limited to, via paracrine factors).

[0032] If the method of the present disclosure reveals that signal transduction from an allele to a downstream factor occurs outside the cell where the allele is expressed, and if a test substance is applied to the female non-human mammal (or cells thereof) and analysis is performed again using the method of the present disclosure, and the signal transduction does not occur between cells (i.e., the downstream factor is no longer detectable), then the applied test substance can be said to be a signal transduction inhibitor. Therefore, the present disclosure also preferably encompasses a method of screening for a signal transduction inhibitor that includes the above steps.

[0033] The test substance is not particularly limited, and compounds, compositions, and gene therapies (including viral vectors) that affect gene expression can be preferably used. Examples of test substances include, but are not limited to, low-molecular-weight compounds, high-molecular-weight compounds (including nucleic acids, antibodies, or fragments thereof), and extracts. The method of administering the test substance is also not particularly limited, and it can be administered to a female non-human mammal by, for example, oral administration, subcutaneous administration, or intravascular administration (particularly intravenous administration). Furthermore, it can be administered to cells (e.g., cultured cells) of a female non-human mammal by adding it to the culture medium.

[0034] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0035] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0036] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0037] Generation of mice with fluorescent protein genes knocked into the X chromosome A reporter gene was inserted into the intron region of the mouse moesin gene (Gene ID: 17698, mRNA: NM_010833.2, Protein: NP_034963.2) as follows. Specifically, the fluorescent protein gene tdTomato was inserted as a reporter gene into the intron between Exon 2 and Exon 3 (corresponding to Exon numbers 3 and 4 in the National Institute on Aging (NIA) information) in the NCBI mouse genome information (NC_000086). The "CAG promoter-tdTomato-pA" construct was introduced into mice by homologous recombination to generate knock-in (KI) mice (X). tdTomato mice) were created.

[0038] Figure 1 shows a schematic diagram of the "CAG promoter-tdTomato-pA." It also shows a schematic diagram of the target allele and knock-in allele. As shown in Figure 1, the "FRT-Neo-FRT" sequence was inserted downstream of the "CAG promoter-tdTomato-pA," and the (PGK)Neo cassette was removed using the Flp-FRT system.

[0039] In the above example, the insertion site for the fluorescent protein gene on the X chromosome was determined to be an intron of the moesin gene. This was determined by searching using the National Center for Biotechnology Information (NCBI) website (https: / / www.ncbi.nlm.nih.gov / ). Approximately 1,500 genes exist on the X chromosome, and a search was conducted to find genes whose insertion would least likely interfere with their original function and which were expressed as ubiquitously as possible, leading to the determination of an intron of the moesin gene as the insertion site. Previous reports have shown that the moesin gene is expressed ubiquitously, and furthermore, it has been reported that knockout mice do not exhibit any phenotype (Non-Patent Document 1, cited above).

[0040] Furthermore, from the viewpoint of stable expression of fluorescent proteins, it is preferable to select homologous recombination as the recombination method because, for example, genome editing tools such as CRISPR / Cas9 and transcription activator-like effector nuclease (TALEN) may result in mismatches of one or several bases, making it difficult to generate KI mice in which two fluorescent proteins behave identically.

[0041] In patients with X-linked hypophosphatemic rickets (XLH), which is inherited as an X-linked dominant disorder, fibroblast growth factor 23 (FGF23) is known to be overexpressed and secreted in bone tissue. Excess FGF23 secreted from bone tissue acts on the kidney, causing phosphorus diuresis, resulting in hypophosphatemic rickets.

[0042] It is also known that X-linked hypophosphatemic rickets (XLH) develops when the PHEX gene located on the X chromosome is inactivated (e.g., by an inactivating mutation). Therefore, the PHEX gene is thought to be a gene that functions to suppress FGF23 expression. However, it was not known how the PHEX gene suppresses FGF23 expression, and the signaling pathway from the PHEX gene to FGF23 was unknown (Figure 2).

[0043] To investigate how the signal is transmitted from the PHEX gene to FGF23, we performed the following study using a female X chromosome model of XLH, which has a PHEX gene mutated on one X chromosome (PHEXmut) and a normal PHEX gene on the other X chromosome (PHEXwt). PHEXmut X PHEXwt Mice were cultured in the same manner as above. tdTomato+ The two X chromosomes of the resulting female mice were designated X1 and X2. X1 carried the normal allele (PHEXwt) and the fluorescent protein (tdTomato) gene, while X2 carried the mutant allele (PHEXmut). tdTomato+ / PHEXwt / X2 tdTomato- / PHEXmut As a control, female mice (X1 tdTomato+ / PHEXwt / X2 tdTomato- / PHEXwt mouse) was created.

[0044] Observation of mouse tissues Frozen sections were prepared from the femurs of the mice (4% PFA perfusion fixation, 10 μm frozen sections). Immunostaining was performed using a green fluorescent dye-labeled anti-FGF23 antibody (Abcam, Goat; polyclonal, RRID: AB_880086) and observed under a fluorescence microscope. DAPI staining images were also obtained. The following describes the reagents and equipment used in this study and the studies described below.

[0045] [Primary antibody] Rat anti-FGF23 Cat. No. MAB26291 R&D systems, Inc., Minneapolis, MN Dilution: 1:100

[0046] rat anti-osterix Cat. No. ab22552 Abcam, Cambridge, UK Dilution: 1:100

[0047] [Secondary antibody] Alexa Fluor 488 conjugated Goat anti-Rat-IgG (H+L) Cat. No. A-11006 Invitrogen Co., Camarillo, CA Dilution: 1:100

[0048] [Encapsulant] VECTASHIELD hard-set mounting medium with DAPI Cat. No. H-1500 Vector Laboratories, Inc. Burlingame, CA

[0049] [microscope] Nikon Eclipse Ni-E microscope, Nikon Instruments Inc. Tokyo, Japan

[0050] [Observation wavelength and filter] Alexa Flour 488 Excitation wavelength: 495 nm, Emission wavelength: 519 nm (Filter cube used: B-2A) tdtomato Excitation wavelength 554 nm, Emission wavelength 581 nm (Filter cube used: G-2A) DAPI Excitation wavelength: 345 nm, Emission wavelength: 455 nm (Filter cube used: DAPI)

[0051] The results of observation of frozen sections of femurs are shown in Figure 3. The upper part of Figure 3 shows a female mouse (X1 tdTomato+ / PHEXwt / X2 tdTomato- / PHEXwt The upper left image shows immunostaining with anti-FGF23 antibody, and FGF23-expressing cells are stained green. The upper right image shows the nuclei stained with DAPI, allowing the location of the cells to be confirmed. A female mouse (X1 tdTomato+ / PHEXwt / X2 tdTomato- / PHEXwt In mice, FGF23 expression was confirmed regardless of the presence or absence of tdTomato expression. This indicates that FGF23 is expressed in all normal bone cells, consistent with previous reports (Nagata Y., Endocrinology 2019;160:1348-1358).

[0052] The bottom row of Figure 3 shows a female mouse (X1 tdTomato+ / PHEXwt / X2 tdTomato- / PHEXmut The normal cells are tdTomato-positive, while cells carrying the pathogenic mutant PHEX gene (pathological cells) are tdTomato-negative. FGF23 is expressed in all bone cells. To confirm the intensity of FGF23 expression in each cell, the exposure time for the lower panel of Figure 3 was shorter than that for the upper panel. Since FGF23 is more strongly expressed in tdTomato-negative cells than in tdTomato-positive cells, it can be concluded that the signaling pathway from the mutant PHEX gene to FGF23 is completed within the tdTomato-negative cells without intercellular communication (Figure 4).

[0053] UMR-106 cells are osteocyte-lineage cultured cells that express Osterix, an osteocyte-specific transcription factor, along with FGF23. When Osterix expression was suppressed in these cells using siRNA (small interfering RNA) against Osterix (Fig. 5, left), FGF23 expression decreased with a delay (Fig. 5, right). These results indicate that Osterix is ​​involved in FGF23 expression, and it is known that the mutant PHEX gene enhances Osterix expression. Osterix is ​​thought to be involved in the mechanism by which the mutant PHEX gene enhances FGF23 expression, but it is unknown whether this signaling pathway is completed intracellularly or mediated by intercellular signaling.

[0054] The upper part of Figure 6 shows a female mouse (X1 tdTomato+ / PHEXwt / X2 tdTomato- / PHEXwt The upper left image shows immunostaining with anti-Osterix antibody, with Osterix-expressing cells appearing green. The upper right image shows the nuclei stained with DAPI, allowing the location of the cells to be confirmed. A female mouse (X1 tdTomato+ / PHEXwt / X2 tdTomato- / PHEXwt In mice, Osterix expression was confirmed regardless of the presence or absence of tdTomato expression, indicating that Osterix is ​​expressed in normal bone cells.

[0055] The bottom row of Figure 6 shows a female mouse (X1 tdTomato+ / PHEXwt / X2 tdTomato- / PHEXmut The cells are tdTomato-positive, while cells carrying the pathogenic mutant PHEX gene (pathological cells) are tdTomato-negative. Osterix is ​​known to be expressed in all bone cells. Therefore, we shortened the exposure time and checked the strength of Osterix expression in each cell. Osterix was strongly expressed in tdTomato-negative cells, which indicates that the signaling pathway from the mutant PHEX gene to Osterix is ​​completed only within tdTomato-negative cells (Figure 4).

[0056] Results of UMR-106 cells and female mice (X1) showing a rickets phenotype tdTomato+ / PHEXwt / X2 tdTomato- / PHEXmut Combined with the results from the mouse model, these findings suggest that the signal transduction pathway from the mutant PHEX gene to increased FGF23 expression is completed within cells harboring the mutant PHEX gene, and that increased Osterix expression is involved in this process. Therefore, suppressing Osterix expression in pathological bone cells is a potential therapeutic target for the treatment of rickets. Furthermore, because the signal transduction pathway from the mutant PHEX gene to increased FGF23 expression is completed within cells harboring the mutant PHEX gene, it is anticipated that therapeutic approaches targeting intercellular signaling will not be feasible. Thus, this method is useful for exploring novel therapies for X-linked dominant genetic disorders.

Claims

1. Signal transduction from an allele protein to a factor (downstream factor) present downstream of the allele protein in signal transduction It is carried out only in cells in which the allele protein is expressed. A method for analyzing When the two X chromosomes of a female non-human mammal are designated as X1 and X2, X1 contains a normal allele and a fluorescent protein gene. The mutant allele on X2 exists, the mutant allele is a gene that exhibits a dominant inherited trait; In female non-human mammalian cells, distinguishably staining the downstream factor and detecting the fluorescent protein; method.

2. The method of claim 1, wherein the downstream factor is a protein (downstream protein) of a gene that is located downstream of the allele protein in signal transduction.

3. the fluorescent protein gene present in X1 is a fluorescent protein gene knocked into X1, The knock-in site is an intron site of a gene on the X chromosome.

3. The method according to claim 1 or 2.

4. The intron site of the gene on the X chromosome into which the fluorescent protein gene is knocked in is the intron site of Moesin. The method of claim 3.

5. The method of claim 3 or 4, wherein the knock-in fluorescent protein gene is a green fluorescent protein gene, a yellow fluorescent protein gene, or a red fluorescent protein gene.

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