JP7852838B2 - Mice with a mutant ALK2 gene - Google Patents

Description

This invention relates to rodents possessing a rodent variant of the ALK2 gene.

Bone morphogenetic proteins (BMPs) are a group of growth factors classified under the Transforming Growth Factor-β (TGF-β) family. BMPs up to BMP15 have been identified, and these, as homodimers or heterodimers, exert diverse physiological activities, including the development and maintenance of musculoskeletal systems such as bone, cartilage, and muscle, and iron metabolism in the liver.

The activity of the TGF-β family, including BMPs, is transmitted by seven transmembrane serine/threonine kinase receptors, classified as type I, called activin-like kinases (ALKs) 1 to ALK7, which are expressed on the cell membrane of target cells. However, the activity of the TGF-β family is modified by type II receptors, which are other transmembrane serine/threonine kinases, and by various other factors, and many aspects of their physiological effects in vivo remain unclear.

ALK2 is a transmembrane serine/threonine kinase receptor that transmits the bone-inducing activity of BMP. ALK2 has been reported to be associated with intractable diseases caused by gene mutations. Examples of diseases caused by such gene mutations include fibrodysplasia ossificans progressive (FOP), in which ectopic ossification occurs throughout the body; diffuse idiopathic skeletal hyperostosis (DISH), in which tendons and ligaments ossify; diffuse intrinsic pontine glioma (DIPG), a type of brain tumor in children; and congenital heart diseases (CHDs).

The physiological roles of BMP and TGF-β family activity, the pathogenesis of related diseases, and the development of therapeutic drugs are primarily studied using mice as a model. However, even when mutations from FOP cases are introduced into mouse ALK2, the mice die immediately after birth, making it impossible to establish a disease model as a clinical line (see, for example, Non-Patent Document 1).

Therefore, there is a strong need to establish a mouse model that can reflect human ALK2-mediated BMP signaling.

Chakkalakal SA, Zhang D, Culbert AL, Convente MR, Caron RJ, Wright AC, Maidment AD, Kaplan FS, Shore EM. An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans progressiva. J Bone Miner Res. 2012 Aug; 27(8):1746-56.

This invention aims to solve the aforementioned problems of the prior art and achieve the following objective: Specifically, it aims to provide a rodent that can be used as an animal model reflecting ALK2-mediated BMP signaling in primates such as humans.

The inventors, through diligent research to achieve the above objective, discovered that among the seven type I transmembrane serine/threonine kinase receptors called ALK1 to ALK7, only ALK2 exhibits a difference in activity between mice and humans. They found that mouse ALK2 has higher activity than human ALK2, and that the main reason for this difference is a single amino acid residue at position 330 in the intracellular kinase domain.

The means to solve the aforementioned problem are as follows:
<1> Possesses a rodent mutant ALK2 gene with a mutation at the position encoding the 330th amino acid of the ALK2 protein,
The aforementioned rodent mutant ALK2 gene is a rodent characterized by a mutation in which the 330th serine in the rodent wild-type ALK2 protein becomes an amino acid selected from the group consisting of proline, asparagine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine.

According to the present invention, the aforementioned problems of the conventional approach can be solved, and a rodent can be provided that can be used as an animal model reflecting ALK2-mediated BMP signaling in primates such as humans.

Figure 1A shows the results of measuring the activity of bone-inductive signals (Smad1/5/9) in Test Example 1. Figure 1B shows the results of measuring the activity of non-osteogenic signals (Smad2/3) in Test Example 1. Figure 2A shows the results of measuring the activity when BMP7 was used in Test Example 2. Figure 2B shows the results of measuring the activity when BMP9 was used in Test Example 2. Figure 3 shows the results of the activity measurement in Test Example 3. Figure 4 shows the results of analyzing heterotopic bone using micro-CT in Test Example 5. Figure 5 shows the results of hematoxylin and eosin staining in Test Example 6. Figure 6 shows the results of measuring the activity in Test Example 7.

(rodent)
The rodents of the present invention (hereinafter sometimes referred to as "ALK2 S330 substituted rodents,""ALK2 S330 mutant rodents,""ALK2 S330P rodents," or "ALK2 S330P mutant rodents") have a rodent mutant ALK2 gene having a mutation at the position encoding the 330th amino acid of the ALK2 protein, and the rodent mutant ALK2 gene includes at least a mutation in which the 330th serine in the rodent wild-type ALK2 protein becomes an amino acid selected from the group consisting of proline, asparagine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine, and may include other mutations as needed.

<ALK2>
ALK2 is a transmembrane serine/threonine kinase receptor that transmits the bone-inducing activity of BMP. ALK2 has been reported to be associated with intractable diseases caused by gene mutations such as FOP, DISH, DIPG, and CHDs.

The amino acid sequence information for ALK2 can be obtained from publicly known databases.
For example, in the database "National Center for Biotechnology Information (NCBI)," human ALK2 can be obtained under the accession numbers NP_001096, monkey ALK2 under NP_001247690.1, mouse ALK2 under NP_001103675.1 (Sequence ID 3) , and rat ALK2 under NP_077812.1.

In the ALK2 amino acid sequence, the 330th amino acid is serine in rodents, while it is proline in primates; the two are different.
As shown in the section [Examples] below, among the seven type I receptors, ALK2 is more active in rodents (mice) than in primates (humans).

The rodents of the present invention possess a rodent mutant ALK2 gene having a mutation at the position encoding the 330th amino acid of the ALK2 protein.
The rodent mutant ALK2 gene contains at least one mutation in which the 330th serine in the rodent wild-type ALK2 protein becomes an amino acid selected from the group consisting of proline, asparagine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine.

As mentioned above, the amino acid at position 330 of the ALK2 protein differs between rodents and primates.
Furthermore, as shown in the [Examples] section below, replacing the 330th proline in human ALK2 with serine from rodents (mouse) increases BMP activity, while replacing the 330th serine in mouse ALK2 with human proline, asparagine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, or valine decreases BMP activity. Therefore, it is thought that the amino acid located at the 330th position of the ALK2 protein controls its activity.
In the rodents of the present invention, the 330th serine in the rodent wild-type ALK2 protein is replaced by an amino acid selected from the group consisting of proline, asparagine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine, thereby reflecting ALK2-mediated BMP signaling in primates. Among these mutations, the mutation in which the 330th serine in the rodent wild-type ALK2 protein is replaced by proline is preferred.

The aforementioned rodents may be heterozygous or homozygous for the rodent variant ALK2 gene, but homozygous is preferred.

The aforementioned rodents can be suitably used as a model that reflects ALK2-mediated BMP signaling in primates. Reflecting ALK2-mediated BMP signaling in primates means that rodents reflect the interaction between ALK2 and its ligand, BMP, in primates.
There are no particular restrictions on the type of BMP mentioned above; it can be selected as appropriate depending on the purpose.

There are no particular restrictions on the species of rodent mentioned above; they can be appropriately selected depending on the purpose. Examples include mice, rats, gerbils, Okinawa mice, Sikkim mice, and Chinese hamsters. Among these, mice and rats are preferred due to their usefulness as experimental animals.

There are no particular restrictions on the primates used; they can be appropriately selected depending on the purpose. Examples include humans and monkeys. Among these, humans are preferred.

As shown in the [Examples] section below, the rodents of the present invention exhibit suppressed ectopic ossification compared to rodents possessing the wild-type ALK2 gene. Furthermore, unlike rodents with the wild-type ALK2 gene, lymphocyte infiltration is observed in the liver.

Other mutations in the aforementioned rodent variant ALK2 gene are not particularly limited and can be appropriately selected depending on the purpose. Examples include gene mutations in diseases caused by gene mutations such as FOP, DISH, DIPG, and CHDs.
Since the rodents of the present invention can reflect ALK2-mediated BMP signaling in primates such as humans, by further introducing mutations of hereditary diseases in primates such as humans, they can be used as models that reflect said hereditary diseases.

The rodents of this invention have a primate-type ALK2 molecule at amino acid position 330, resulting in lower activity compared to wild-type rodent ALK2. This allows them to reflect ALK2-mediated BMP signaling in primates. Therefore, it is possible to establish disease models for genetic diseases that could not be established using conventional mice.

The method for producing rodents according to the present invention is not particularly limited, and known methods can be appropriately selected. For example, one method involves introducing mutations using genome editing technology with Cas9 protein, guide RNA (a sequence within the rodent ALK2 gene), and donor oligo DNA (derived from the rodent ALK2 gene sequence).

For example, when using mice as the rodents, a method can be used in which the guide RNA is, for example, one whose target sequence is the sequence represented by Sequence ID No. 1, and the donor oligo DNA is the sequence represented by Sequence ID No. 2. In this example, a total of four base mutations are introduced at the chr2: 58,462,951-58,462,959 locus (on the 8th exon of the ALK2 gene). The amino acid sequences at positions 331 and 332 remain Ala-Ile due to synonymous substitution, and Ser at position 330 is replaced with Pro.
<Target sequences of guide RNA>
5'-CAGATCTCGATTGGGCAATTGGcgg-3' (Sequence ID 1: chr2: 58,462,936-58,462,958)
The lowercase part (cgg) in sequence number 1 indicates the PAM sequence.
<Donor OligoDNA (ssDNA)>
5'-GGTTAGCTGCCTTCGGATTGTACTGTCCATAGCCAGCGGCCTTGCCCATTTGCACATAGAGATATTTGGGACCCAAGGGAAG C C T GC T AT C GCCCATCGAGATCTGAAGAGGCAAAAAACATCCTGGTGAAGAAGA-3' (SEQ ID NO: 2)
The underlined portion in Sequence ID No. 2 indicates the location of the mutation.

The rodents of this invention enable the analysis of the physiological roles of ALK2-mediated BMP signaling in primates, particularly in the musculoskeletal system and liver. Furthermore, they are expected to contribute to elucidating the pathogenesis of various diseases caused by ALK2 gene mutations, and to the development of therapeutic and preventive drugs for these diseases and fractures. They can also be used as experimental animals for pathological models of ectopic ossification and DIPG caused by ALK2 genetic mutations, such as FOP and DISH, and for analyzing ALK2 signaling.

The following describes examples of tests conducted using the present invention, but the present invention is not limited to these examples.

(Example 1 of the experiment: Among the seven types of type I receptors, ALK2 shows higher activity in mice than in humans.)
We measured the activity of intracellular signaling mediated by seven type I receptors using a BMP-specific luciferase reporter.
Specifically, HEK293A cells were seeded in a 96-well white plate for luciferase assays (Greiner) at a density of 1 × ^10⁴ cells/well, and cultured overnight in DMEM medium containing 10% FBS at 37°C and 5% _CO₂ . The following day, pcDEF3/HitALK1(WT)-V5-His (human wild type, sometimes referred to as "ALK1"), pcDEF3/HitALK2(WT)-V5-His (human wild type, sometimes referred to as "ALK2"), pcDEF3/HitALK3(WT)-V5-His (human wild type, sometimes referred to as "ALK3"), pcDEF3/HitALK4(WT)-V5-His (human wild type, sometimes referred to as "ALK4"), pcDEF3/HitALK5 (WT)-V5-His (human wild type, sometimes referred to as "ALK5"), pcDEF3/hitoALK6(WT)-V5-His (human wild type, sometimes referred to as "ALK6"), pcDEF3/hitoALK7(WT)-V5-His (human wild type, sometimes referred to as "ALK7"), or pcDEF3 (used to adjust the total amount of plasmids to a certain level; sometimes referred to as "mock") and pGL4.26/IdlWT4F-luc(Genes Cells, 7, 949 (2002); Biochem Biophysis Res Commun, 407, 213 (2011)) and phRL-SV40 (Promega) were installed using Lipofectamine 2000 (Thermo Fisher Scientific). After 2.5 hours, the following were administered: 0.5 ng/mL of BMP9 (manufactured by Peprotech, sometimes referred to as "B9"), 10 ng/mL of BMP7 (manufactured by Milltenyi Biotec, sometimes referred to as "B7"), 10 ng/mL of BMP2 (manufactured by Corefront, sometimes referred to as "B2"), 10 ng/mL of Activin A (manufactured by Peprotech, sometimes referred to as "ActA"), 1 ng/mL of TGF-β1 (manufactured by Peprotech, sometimes referred to as "Tb1"), 10 ng/mL of GDF5 (manufactured by Peprotech, sometimes referred to as "GDF5"), or 10 ng/mL of Activin The cells were replaced with OPTI-MEM I (Thermo Fisher Scientific) containing B (Peprotech, sometimes referred to as "ActB") and incubated overnight. The following day, the luciferase activity of Firefly and Renilla was measured using a Dual-Glo Luciferase Assay System (Promega) and a plate reader GENios (TECAN).

Figure 1A shows the results of measuring the activity of bone-inducible signals (Smad1/5/9), and Figure 1B shows the results of measuring the activity of non-bone-inducible signals (Smad2/3). In Figures 1A and 1B, the items on the horizontal axis represent mocks or each type I receptor, and the bar graphs for each item show the results for mocks, humans, and mice, respectively, from left to right.
As shown in Figures 1A and 1B, ALK2, which is involved in the transmission of bone-inducible signals, was found to be several times more active in mice than in humans.

(Test example 2: Amino acid 330 of ALK2 controls its activity)
We measured the activity of intracellular signaling mediated by ALK2 using a BMP-specific luciferase reporter.
Specifically, HEK293A cells were seeded in a 96-well white plate for luciferase assays (Greiner) at a density of 1 × ^10⁴ cells/well, and cultured overnight in DMEM medium containing 10% FBS at 37°C and 5% _CO₂ . The following day, pcDEF3/hitoALK2(WT)-V5-His (human wild type, sometimes referred to as "human"), pcDEF3/hitoALK2(P330S)-V5-His (human wild type modified to mouse type by substituting proline at position 330 with serine, sometimes referred to as "hP330S"), and pcDEF3/mouseALK2(WT)-V5-His (mouse wild type, sometimes referred to as "mouse") were cultured. It may be called, ), pcDEF3/mouseALK2(S330P)-V5-His (modified to human form by substituting serine at position 330 with proline in the mouse wild type, sometimes referred to as "mS330P"), or pcDEF3 (used to adjust the total amount of plasmid to a certain level, sometimes referred to as "mock"), and pGL4.26/IdlWT4F-luc (Genes Cells, 7, 949 (2002); Biochem Biophys Res Commun, 407, 213 (2011)) and phRL-SV40 (Promega) were introduced using Lipofectamine 2000 (Thermo Fisher Scientific). After 2.5 hours, the culture was changed to OPTI-MEM I (Thermo Fisher Scientific) containing 10 ng/mL BMP7 (Miltenyi Biotec) or 0.5 ng/mL BMP9 (Peprotech), and cultured overnight. The following day, the luciferase activity of Firefly and Renilla was measured using a Dual-Glo Luciferase Assay System (Promega) and a GENios plate reader (TECAN).

Figure 2A shows the results when using BMP7, and Figure 2B shows the results when using BMP9. In Figures 2A and 2B, the horizontal axis items represent the expression vectors used, and the bar graphs for each item show the results when using control, BMP7, or BMP9, from left to right.
As shown in Figures 2A and 2B, it was confirmed that replacing the 330th proline in human ALK2 with a mouse-type serine increased BMP activity, while replacing the 330th serine in mouse ALK2 with a human-type proline decreased BMP activity.

(Test example 3: ALK2 amino acid 330 regulates ligand-dependent activity)
We measured ligand-dependent activity mediated by ALK2 using a BMP-specific luciferase reporter.
Specifically, HEK293A cells were seeded in a 96-well white plate for luciferase assays (Greiner) at a density of 1 × ^10⁴ cells/well, and cultured overnight in DMEM medium containing 10% FBS at ₃₇ °C and 5% CO₂. The following day, pcDEF3/hitoALK2(WT)-V5-His (human wild type, sometimes referred to as "hA2WT"), pcDEF3/hitoALK2(P330S)-V5-His (modified to the mouse type by substituting proline at position 330 of the human wild type with serine, sometimes referred to as "hA2(P330S)"), or pcDEF3 (used to adjust the total amount of plasmid to a certain level, sometimes referred to as "mock") and pGL4.26/IdlWT4F-luc (Genes Cells, 7, 949 (2002); Biochem Biophys Res Commun, 407, 213) (2011)) and phRL-SV40 (manufactured by Promega) were installed using Lipofectamine 2000 (manufactured by Thermo Fisher Scientific). After 2.5 hours, the culture was replaced with OPTI-MEM I (Thermo Fisher Scientific) containing 1 ng/mL, 10 ng/mL, or 100 ng/mL of BMP2 (Corefront), BMP4 (R&D), BMP5 (Peprotech), BMP6 (Peprotech), BMP7 (Mitenyi Biotec), BMP9 (Peprotech), BMP10 (Mitenyi Biotec), GDF5 (Peprotech), or Activin B (Peprotech), and the culture was incubated for another night. The following day, the luciferase activity of Firefly and Renilla was measured using a Dual-Glo Luciferase Assay System (Promega) and a GENios plate reader (TECAN).

The results are shown in Figure 3. In Figure 3, the horizontal axis items represent the various BMPs, GDF5, and Activin B (Act B) used, and "1, 10, 100" within each item represent the amount of each BMP, GDF5, and Activin B (Act B) used. The bar graphs for each amount of use show the results when using Mock, hA2WT, and hA2 (P330S), in order from left to right.
As shown in Figure 3, in BMP2, 4, and 10, both the wild type (hA2WT) and the mutant type (hA2(P330S)) showed decreased activity. On the other hand, in BMP5, 6, 7, and 9, the mutant type increased activity. In the case of Activin B, the wild type did not respond, but the mutant type did.
These results suggest that mouse ALK2 does not accurately reflect the activity of human ALK2. Therefore, conventional mouse models that incorporate mutations for human genetic diseases are likely not accurate models of human genetic diseases.

BMP7, discovered as a bone-inducing factor and whose clinical application is being developed, is thought to be one of the BMPs that exerts its biological activity using ALK2 as a receptor. It has long been known that BMPs exhibit strong bone-inducing activity in rodents, while their activity is weak in primates, including humans, but the mechanism remains unclear. Based on the above results, it is possible that a difference in residue 330 of ALK2 influences the bone-inducing activity of BMPs such as BMP7.

(Example 4: Establishment of ALK2 S330P mice through genetic modification)
We established the ALK2 S330P mutant mouse (a mouse in which the 330th Ser in the mouse ALK2 gene is replaced with a human-type Pro) by introducing mutations using genome editing technology (CRISPAR/Cas9) with Cas9 protein, guide RNA (mouse ALK2 gene sequence), and donor oligo DNA (derived from the mouse ALK2 gene sequence). In the ALK2 S330P mouse, a total of 4 base pairs were introduced at the chr2: 58,462,951–58,462,959 locus (on exon 8 of the ALK2 gene). As a result, the 330th Ser in the ALK2 protein was replaced with Pro (the amino acid sequences at positions 331 and 332 remained Ala-Ile due to synonymous substitution).
The established mice showed no macroscopic abnormalities and were able to maintain normal breeding practices.
<Target sequences of guide RNA>
5'-CAGATCTCGATTGGGCAATTGGcgg-3' (Sequence ID 1: chr2: 58,462,936-58,462,958)
The lowercase part (cgg) in sequence number 1 indicates the PAM sequence.
<Donor OligoDNA (ssDNA)>
5'-GGTTAGCTGCCTTCGGATTGTACTGTCCATAGCCAGCGGCCTTGCCCATTTGCACATAGAGATATTTGGGACCCAAGGGAAG C C T GC T AT C GCCCATCGAGATCTGAAGAGGCAAAAAACATCCTGGTGAAGAAGA-3' (SEQ ID NO: 2)
The underlined portion in Sequence ID No. 2 indicates the location of the mutation.

(Test example 5: Ectopic ossification is suppressed in ALK2 S330P mice)
The effect of ALK2 S330P on ectopic ossification was analyzed using an ectopic ossification model induced by BMP transplantation. BMP7 pellets were prepared by impregnating 2.5 μg of BMP7 (Milteney) into a 4 mm diameter circular piece of CollaTape (Zimmer Dental) and freeze-drying it. ALK2 WT/WT mice (wild-type), ALK2 S330P/WT mice (heterozygous), and ALK2 S330P/S330P mice (homozygous) were subjected to a 2% isoflurane inhalation anesthesia, and the thigh was incised, with the BMP7 pellets transplanted into the skeletal muscle. Three weeks after transplantation, ectopic bone was analyzed using micro-CT (CosmoScanGX).

The results are shown in Figure 4. In Figure 4, the top row (WT/WT) shows the results for three wild-type individuals, the middle row (S330P/WT) shows the results for three heterozygous individuals, and the bottom row shows the results for three homozygous individuals. In Figure 4, the white arrows indicate ectopic bone.
As shown in Figure 4, heterozygotes and homozygotes were found to have smaller ectopic bone size compared to the wild type. This result is thought to reflect the decrease in activity observed in the in vitro study described above when serine at position 330 in mouse ALK2 was replaced with proline.

(Test Example 6: Phenotypes in the liver of ALK2 S330P mice)
Liver tissue samples were collected from ALK2 WT/WT mice (wild-type), ALK2 S330P/WT mice (heterozygous), and ALK2 S330P/S330P mice (homozygous). These samples were fixed in 4% paraformaldehyde phosphate buffer (Nacalai Tesque) for two days, dehydrated with ethanol, replaced with xylene, and embedded in paraffin. Paraffin sections were prepared using a rotary microtome (Leica), stained with hematoxylin and eosin according to standard procedures, and mounted with coverslips to prepare tissue specimens. The tissue specimens were observed using a BZ-9000 (Keyence).

The results are shown in Figure 5. In Figure 5, the top row (WT/WT) shows the results for three wild-type individuals, the middle row (S330P/WT) shows the results for three heterozygous individuals, and the bottom row shows the results for three homozygous individuals.
As shown in Figure 5, lymphocyte infiltration was observed in the liver in both heterozygous and homozygous individuals.

The results of Test Examples 5 and 6 suggest that ALK2 is an important receptor for BMP7-mediated bone induction activity, and also suggest that ALK2 may have unknown physiological effects in tissues such as the liver.

(Test example 7: Substitution of amino acid 330 in mouse ALK2)
The 330th amino acid (serine) in mouse wild-type ALK2 was substituted with the following amino acid, and the activity of intracellular signaling mediated by ALK2 was measured using a BMP-specific luciferase reporter in the same manner as in Test Example 2. The same test was also performed for mouse wild-type ALK2 without the substitution of the 330th amino acid.
<Substituted amino acids>
(1) Pro
(2) Ala
(3) Arg
(4) Asn
(5) Asp
(6) Cys
(7) Gln
(8) Glu
(9) Gly
(10) His
(11) Ile
(12) Leu
(13) Lys
(14) Met
(15) Phe
(16) Thr
(17) Trp
(18) Tyr
(19) Val

Specifically, HEK293A cells were seeded in a 96-well white plate for luciferase assays (Greiner) at a density of 1 × ^10⁴ cells/well, and cultured overnight in DMEM medium containing 10% FBS at 37°C and 5% _CO₂ . The following day, pcDEF3/mouse ALK2(S330 substitution)-V5-His (mouse wild type with serine at position 330 substituted with the above amino acid), pcDEF3/mouse ALK2(WT)-V5-His (mouse wild type, serine at position 330), or pcDEF3 (used to adjust the total amount of plasmid to a certain level; sometimes referred to as a "mock"), along with pGL4.26/IdlWT4F-luc (Genes Cells, 7, 949 (2002); Biochem Biophys Res Commun, 407, 213 (2011)), and phRL-SV40 (Promega) were mixed with Lipofectamine 2000 (Thermo Luciferase was introduced using a Fisher Scientific instrument. After 2.5 hours, the cells were replaced with OPTI-MEM I (Thermo Fisher Scientific) containing 10 ng/mL of BMP7 (Miltenyi Biotec), and incubated overnight. The following day, the luciferase activity of Firefly and Renilla was measured using a Dual-Glo Luciferase Assay System (Promega) and a GENios plate reader (TECAN).

The results are shown in Figure 6. In Figure 6, the horizontal axis represents the expression vector used. Except for the mock expression, the values are represented by the 330th amino acid in mouse ALK2.
As shown in Figure 6, it was confirmed that BMP activity decreased when the 330th serine in mouse ALK2 was replaced with proline, asparagine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine.

The results above suggest that the rodent of the present invention, which contains a mutation in the wild-type rodent ALK2 protein in which the 330th serine is an amino acid selected from the group consisting of proline, asparagine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine, has the potential to be used as an in vivo model that reflects primate ALK2 with weak BMP activity. A rodent model that reflects BMP signaling in primates such as humans will enable the analysis of the physiological roles of ALK2-mediated BMP signaling in the musculoskeletal system and liver in rodents, and is expected to have applications in elucidating the pathogenesis of various diseases caused by ALK2 gene mutations, as well as in the development of therapeutic and preventive drugs for these diseases and fractures.

Examples of embodiments of the present invention include the following:
<1> Possesses a rodent mutant ALK2 gene with a mutation at the position encoding the 330th amino acid of the ALK2 protein,
The aforementioned rodent mutant ALK2 gene is a rodent characterized by a mutation in which the 330th serine in the rodent wild-type ALK2 protein becomes an amino acid selected from the group consisting of proline, asparagine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine.
<2> The rodents described in <1> above are a model that reflects ALK2-mediated BMP signaling in primates.
<3> A rodent as described in any of <1> to <2> above, which is either a mouse or a rat.
<4> A rodent according to any of <1> to <3> above, in which heterotopic ossification is suppressed.
<5> A rodent according to any of <1> to <4> above, in which lymphocytes are infiltrated in the liver.

Claims (4)

A mouse having a mutant ALK2 gene with a mutation at the position encoding the 330th amino acid of the wild-type ALK2 protein represented by Sequence ID No. 3 ,
The mutant ALK2 gene is characterized by a mouse in which the 330th serine in the wild-type ALK2 protein represented by Sequence ID No. 3 is replaced with an amino acid selected from the group consisting of proline, asparagine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine. The mouse according to claim 1, which is a model that reflects ALK2-mediated BMP signaling in primates. A mouse according to claim 1 or 2, wherein ectopic ossification is suppressed. A mouse according to any one of claims 1 to 3, wherein lymphocytes are infiltrated in the liver.