Construction method and application of transgenic animal model with osteoclast specific deletion

Abstract

The invention provides a construction method and application of a transgenic animal model with osteoclast specific deletion, and belongs to the technical field of gene engineering. The invention provides a method for constructing an animal model of an osteoclast expression diphtheria toxin receptor, and particularly provides a method for constructing the animal model of the osteoclast expression diphtheria toxin receptor by using a Cre-loxP method. According to the method, the diphtheria toxin receptor is specifically expressed in the osteoclast, and the osteoclast can be specifically deleted by injecting the diphtheria toxin. The animal model of the osteoclast expression diphtheria toxin receptor prepared by the method can stably inherit after being established, is economical and reliable, can provide important theoretical and practical guidance for bone tumor treatment, also provides powerful animal model support for research of bone tumor growth correlation, and has a wide application prospect. Technical conditions are provided for screening, developing, preventing or treating medicines for bone tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a transgenic animal model with osteoclast specific deletion and its application. Background Art

[0002] Osteoclasts are multinuclear giant cells derived from hematopoietic stem cells that complete the fusion and differentiation of mononuclear precursor cells through the RANKL / RANK / OPG signaling pathway. Their core function depends on the special structure of the "ruffled border", which actively secretes H through the proton pump. + and Cl - , forming a local acidic microenvironment at the bone matrix interface. This process not only dissolves hydroxyapatite crystals but also releases Ca 2+ , while activating osteolytic enzymes such as cathepsin K (CTSK), which specifically degrade organic components such as type I collagen, and jointly mediate the bone resorption process.

[0003] The maintenance of bone homeostasis depends on a dynamic balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Recent studies have shown that abnormal osteoclast activation can lead to an imbalance in bone resorption and formation, becoming a key pathological mechanism in metabolic bone diseases such as osteoporosis (OP) and rheumatoid arthritis (RA), as well as bone metastasis of malignant tumors. For example, osteoclast activity can increase 3-5 times in postmenopausal osteoporosis patients, and osteoclast density in the metastatic bone tumor microenvironment is significantly positively correlated with the degree of osteolysis.

[0004] Current strategies for regulating osteoclasts mainly include: (1) using bisphosphonates to inhibit osteoclast activity; (2) downregulating the expression of key genes (such as CTSK and TRAP) through RNA interference technology; and (3) conditional gene knockout using transgenic animal models (such as CTSK-Cre). However, existing methods have significant limitations: drug intervention lacks cell specificity and is prone to drug resistance; gene silencing technology is difficult to achieve complete knockout; and the traditional Cre-loxP system is limited by the recombinase activity threshold and cannot achieve efficient deletion of mature osteoclasts. No technology has been reported to specifically delete osteoclasts. Summary of the Invention

[0005] In response to the defects in the prior art, the purpose of the present invention is to provide a method for constructing an animal model in which osteoclasts express diphtheria toxin receptors. The constructed animal model in which osteoclasts express diphtheria toxin receptors can be further injected with diphtheria toxin to obtain a transgenic animal model with osteoclast-specific deletion, thereby providing technical conditions for screening, developing, preventing or treating bone tumor drugs.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for constructing an animal model in which osteoclasts express diphtheria toxin receptor, comprising the following steps:

[0008] The gene expressing the simian diphtheria toxin receptor is inserted into osteoclasts, so that the gene of the simian diphtheria toxin receptor is specifically expressed in osteoclasts, thereby obtaining an animal model in which osteoclasts express the diphtheria toxin receptor.

[0009] Preferably, the species of animal comprises a mammal resistant to diphtheria toxin.

[0010] The present invention provides a method for constructing an animal model of osteoclasts expressing diphtheria toxin receptor using the Cre-loxP method, comprising the following steps:

[0011] The constructed Rosa26iDTR flox / flox Animals and Carrying Ctsk Cre+ / - Crossing the tool animal with the parent to obtain the F0 generation; the Cre gene carried by the tool animal is inserted before the 3'UTR of the Ctsk gene;

[0012] Screening F0 generation genotype is Ctsk Cre+ / - Rosa26iDTR flox / + The animal model is one in which osteoclasts express diphtheria toxin receptor.

[0013] Preferably, the genotype obtained by screening is Ctsk Cre+ / - Rosa26iDTR flox / + The F0 generation of animals also includes:

[0014] The genotype is Ctsk Cre+ / - Rosa26iDTR flox / + Animals with the genotype Ctsk Cre+ / - Rosa26iDTR flox / + The animals were hybridized to obtain the F1 generation; the F1 generation genotype was screened to be Ctsk Cre+ / - Rosa26iDTR flox / + and Ctsk Cre+ / - Rosa26iDTR flox / flox The animal model is one in which osteoclasts express diphtheria toxin receptor.

[0015] Preferably, the F0 generation screening and / or the F1 generation screening comprises performing PCR amplification;

[0016] PCR amplification was performed using the primer pair of Rosa26-iDTR-878bp-F shown in SEQ ID No. 7 and the nucleotide sequence of Rosa26-iDTR-878bp-R shown in SEQ ID No. 8, and a band appeared; and PCR amplification was performed using the primer pair of Ctsk-e(2A-Cre-Wpre-pA)1-403bp-F shown in SEQ ID NO. 3 and the nucleotide sequence of Ctsk-e(2A-Cre-Wpre-pA)1-403bp-R shown in SEQ ID NO. 4, and a band appeared, thereby obtaining an animal model in which osteoclasts express diphtheria toxin receptor.

[0017] Preferably, the primer pair used for screening further comprises Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-F having a nucleotide sequence as shown in SEQ ID NO.1 and Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-R having a nucleotide sequence as shown in SEQ ID NO.2;

[0018] The nucleotide sequence of Rosa26-iDTR-WT-487bp-F is shown in SEQ ID No. 5, and the nucleotide sequence of Rosa26-iDTR-WT-487bp-R is shown in SEQ ID No. 6.

[0019] Preferably, when identifying whether the Ctsk gene or the Ctsk-e(2A-Cre-Wpre-pA)1 gene is inserted, if only Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-F and Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-R can amplify a 442bp band, it indicates that the mouse is a wild-type mouse and no exogenous Ctsk-e(2A-Cre-Wpre-pA)1 gene is inserted; if Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-F and Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-R can amplify a 442bp band, Ctsk-e(2A-Cre-Wpre-pA)1 is inserted. If both Ctsk-e(2A-Cre-Wpre-pA)1-403bp-F and Ctsk-e(2A-Cre-Wpre-pA)1-403bp-R can be amplified to obtain a 403bp band, it indicates that the mouse is a heterozygous mouse, with the exogenous Ctsk-e(2A-Cre-Wpre-pA)1 gene inserted into one chromosome and the exogenous Ctsk-e(2A-Cre-Wpre-pA)1 gene not inserted into one chromosome; if only Ctsk-e(2A-Cre-Wpre-pA)1-403bp-F and Ctsk-e(2A-Cre-Wpre-pA)1-403bp-R can be amplified to obtain a 403bp band, it indicates that the mouse is a homozygous mouse, with the exogenous Ctsk-e(2A-Cre-Wpre-pA)1 gene inserted into both chromosomes;

[0020] In identifying whether the Rosa26iDTR gene is inserted, if only Rosa26-iDTR-WT-487bp-F and Rosa26-iDTR-WT-487bp-R can amplify a 487bp band, it indicates that the mouse is a wild-type mouse and no exogenous Rosa26iDTR gene is inserted; if Rosa26-iDTR-WT-487bp-F and Rosa26-iDTR-WT-487bp-R can amplify a 487bp band, Rosa26-iDTR-WT-487bp-R can amplify a 487bp band. -F and Rosa26-iDTR-878bp-R can amplify an 878bp band, indicating that the mouse is a heterozygous mouse, with the exogenous Rosa26iDTR gene inserted into one chromosome and the exogenous Rosa26iDTR gene not inserted into the other chromosome; if only Rosa26-iDTR-878bp-F and Rosa26-iDTR-878bp-R can amplify an 878bp band, it indicates that the mouse is a homozygous mouse, with the exogenous Rosa26iDTR gene inserted into both chromosomes.

[0021] The present invention provides a method for constructing a transgenic animal model with osteoclast-specific deletion, comprising the following steps:

[0022] The animal model expressing diphtheria toxin receptor in osteoclasts constructed by the method described in the above technical solution is injected with diphtheria toxin to obtain a transgenic animal model with osteoclast-specific deletion.

[0023] The present invention provides an animal model in which osteoclasts express a diphtheria toxin receptor and / or a transgenic animal model with osteoclast-specific deletion for use in screening or developing drugs for treating bone tumors. The animal model in which osteoclasts express a diphtheria toxin receptor is obtained using the construction method described in the above technical solution or is constructed using the method described in the above technical solution; the transgenic animal model with osteoclast-specific deletion is constructed using the construction method described in the above technical solution.

[0024] The present invention provides use of a preparation for specifically deleting osteoclasts in preparing a medicine for preventing and / or treating bone tumors.

[0025] Beneficial effects of the present invention:

[0026] The present invention provides a method for constructing an animal model in which osteoclasts express a diphtheria toxin receptor, comprising the following steps: inserting a gene expressing a simian diphtheria toxin receptor into osteoclasts, causing the gene to be specifically expressed in osteoclasts, and thereby obtaining an animal model in which osteoclasts express the diphtheria toxin receptor. The present invention causes the simian diphtheria toxin receptor to be expressed in osteoclasts. Expression of the simian diphtheria toxin receptor on the cell surface allows diphtheria toxin to bind and subsequently undergo receptor-mediated endocytosis. After entering the cytoplasm, the diphtheria toxin inactivates the eukaryotic cell's peptide elongation factor-2 (EF-2), leading to termination of protein synthesis and apoptosis of the target cells. This allows the construction of a transgenic animal model with an osteoclast-specific deletion. The resulting osteoclast-specific deletion transgenic animal model is characterized by high reliability.

[0027] The present invention provides a method for constructing an animal model in which osteoclasts express a diphtheria toxin receptor using the Cre-loxP method. The method utilizes the Cre-loxP technology to specifically insert a gene expressing a diphtheria toxin receptor into osteoclasts, thereby causing the diphtheria toxin receptor to be specifically expressed in osteoclasts. The animal model in which osteoclasts express a diphtheria toxin receptor can be specifically deleted by injecting diphtheria toxin. The animal model in which osteoclasts express a diphtheria toxin receptor prepared by this method can be stably inherited after establishment, and is economical and reliable. This transgenic mouse model has broad application prospects and market value, and can provide important theoretical and practical guidance for the treatment of bone tumors. It also provides powerful animal model support for research related to bone tumor growth, and provides technical conditions for screening, developing, preventing or treating drugs for bone tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 The parental genotype is Ctsk Cre+ / - Figure 1 shows the PCR identification results of mice;

[0030] Figure 2 The parental genotype is Rosa26iDTR flox / flox Figure 1 shows the PCR identification results of mice;

[0031] Figure 3 For F0 generation Ctsk Cre+ / - Rosa26iDTR flox / + Agarose gel electrophoresis identification results of mice;

[0032] Figure 4 For F1 generation Ctsk Cre+ / - Rosa26iDTR flox / + Mice and Ctsk Cre+ / - Rosa26iDTR flox / flox Agarose gel electrophoresis identification results of mice;

[0033] Figure 5 Ctsk in Example 2 Cre+ / - Rosa26iDTR flox / flox TRAP staining results of osteoclasts in the femur of mice after injection of diphtheria toxin;

[0034] Figure 6This is a graph showing the inhibitory effect of osteoclast ablation on tumor growth in the MC38 bone tumor model in Example 3;

[0035] Figure 7 This is a graph showing the inhibitory effect of osteoclast ablation on tumor growth in the B16 bone tumor model in Example 4. DETAILED DESCRIPTION

[0036] The present invention provides a method for constructing an animal model in which osteoclasts express diphtheria toxin receptor, comprising the following steps:

[0037] The gene expressing the simian diphtheria toxin receptor is inserted into osteoclasts, so that the gene of the simian diphtheria toxin receptor is specifically expressed in osteoclasts, thereby obtaining an animal model in which osteoclasts express the diphtheria toxin receptor.

[0038] The present invention targets the simian diphtheria toxin receptor gene and introduces it into osteoclasts, where it is expressed in osteoclasts but not in other animal cells. The method for expressing the simian diphtheria toxin receptor in osteoclasts is not particularly limited and can be accomplished using conventional methods in the art. In the present invention, the animal species preferably includes mammals resistant to diphtheria toxin, more preferably mice and / or rats.

[0039] The present invention enables the expression of monkey diphtheria toxin receptors in osteoclasts. The expression of monkey diphtheria toxin receptors on the cell surface allows diphtheria toxin to bind and subsequently undergo receptor-mediated endocytosis. After entering the cytoplasm, diphtheria toxin inactivates the peptide chain elongation factor-2 (EF-2) of eukaryotic cells, leading to the termination of protein synthesis and thus target cell apoptosis, thereby constructing a transgenic animal model with osteoclast-specific deletion.

[0040] The present invention provides a method for constructing an animal model of osteoclasts expressing diphtheria toxin receptor using the Cre-loxP method, comprising the following steps:

[0041] The constructed Rosa26iDTR flox / flox Animals and Carrying Ctsk Cre+ / - Crossing the tool animal with the parent to obtain the F0 generation; the Cre gene carried by the tool animal is inserted before the 3'UTR of the Ctsk gene;

[0042] Screening F0 generation genotype is Ctsk Cre+ / - Rosa26iDTR flox / + The animal model is an animal model in which osteoclasts express diphtheria toxin receptor. flox / + Indicates heterozygote, Rosa26iDTR flox / flox Indicates homozygote.

[0043] The following uses the construction of a mouse model as an example to illustrate the construction of an animal model. In the following technical solution description, the Ctsk gene and the Ctsk-e(2A-Cre-Wpre-pA)1 gene have the same meaning.

[0044] The present invention is based on the constructed Rosa26iDTR flox / flox Animals and Carrying Ctsk Cre+ / - The tool animal is used as a parent for hybridization to obtain the F0 generation; the Cre gene carried by the tool animal is inserted before the 3'UTR of the Ctsk gene. The present invention has no special restrictions on the construction method of the parent, and conventional construction methods in the art can be used. In the present invention, the Rosa26iDTR flox / flox The animals preferably include Rosa26-iDTR mice; the expression of the simian diphtheria toxin receptor (DTR) of the Rosa26-iDTR mice is blocked by the upstream loxP-Stop-loxP frame; the Rosa26-iDTR mice are preferably purchased from Suzhou Saiye Biotechnology Co., Ltd. In the present invention, the genotype of the Rosa26-iDTR mice can be preferably expressed as Rosa26iDTR flox / flox In the present invention, Ctsk Cre+ / - The tool animal preferably includes Ctsk-e (2A-Cre-Wpre-pA) 1 mouse; the Ctsk-e (2A-Cre-Wpre-pA) 1 mouse has a 2A-Cre-Wpre-polyA co-expression structure inserted before the 3'UTR of the mouse endogenous Ctsk gene; the genotype of the Ctsk-e (2A-Cre-Wpre-pA) 1 mouse can be preferably expressed as Ctsk Cre+ / - , which can also be expressed as Ctsk-e(2A-Cre-Wpre-pA)1 + / - The Ctsk-e(2A-Cre-Wpre-pA)1 mice were preferably purchased from Shanghai Model Organisms Science Co., Ltd. In the present invention, the Rosa26iDTR flox / flox Mice and Ctsk Cre+ / - The genetic tool mice were all of SPF grade C57BL / 6 genetic background.

[0045] The present invention preferably performs adaptive breeding on the above-mentioned parents before hybridization; more preferably, hybridization is performed when the mice are 6 to 8 weeks old. The present invention preferably selects sexually mature healthy mice for hybridization. The selection conditions described in the present invention preferably include: 1) Body shape: strong physique, well-proportioned body, strong limbs, no abnormal phenomena such as tilted head; 2) Coat: smooth and shiny coat, close to the body and not easy to fall off, without external injuries; 3) Eyes: bright and bright eyes, no secretions from the corners of the eyes, no abnormal conditions such as cataracts and blindness; 4) Breathing: clean nostrils and normal breathing; 5) Feces: clean anus and uniform fecal pellets; 6) Genitals: when selecting suitable breeding mice, it is also necessary to observe whether the male mice have lost their genitals, and the female mice need to be checked for double vaginas / vaginal closure; 7) Behavior: quick reaction, natural and lively movements, good appetite, no abnormal behaviors such as circling and sluggishness.

[0046] After selecting mice that meet the conditions, the present invention hybridizes the corresponding mice. When hybridizing, the present invention preferably cages different mice together; the time for cage combination is preferably from dusk to the early morning of the next day. When hybridizing, the present invention preferably has a male-female mating ratio of 1:2 between the two types of mice. The present invention preferably determines whether mating is successful by detecting whether a vaginal plug is formed in the female mouse. The present invention preferably feeds successfully mated female mice separately. In the feeding process of the present invention, it is preferred to add sufficient feed and water, ensure that the bedding is clean, and regularly observe the mice for delivery; the successfully mated mice preferably give birth about 21 days after successful mating to obtain offspring mice, i.e., F0 generation mice.

[0047] The present invention divides the genotype into Ctsk-e(2A-Cre-Wpre-pA)1 + / - and Rosa26-iDTR + / + When mice are used as parents for hybridization, the F0 generation of mice preferably produces two genotype mice, namely Ctsk-e(2A-Cre-Wpre-pA)1 + / - / Rosa26-iDTR + / - Mouse, Ctsk-e(2A-Cre-Wpre-pA)1 - / - / Rosa26-iDTR + / - mouse.

[0048] After obtaining F0 generation mice, the present invention preferably identifies the F0 generation mice. The present invention preferably identifies the F0 generation mice after weaning, that is, 18 to 28 days after birth, more preferably 21 days. The present invention preferably identifies the F0 generation mice by taking ear tissue samples from the mice for identification. In the present invention, the identification method preferably includes PCR identification. In the present invention, the F0 generation screening method preferably includes using a primer pair designed by Ctsk to perform PCR amplification, and the genotype of the amplified band is Ctsk Cre+ / - Rosa26iDTRflox / + In the present invention, the Ctsk preferably includes Ctsk Cre+ / + and Ctsk Cre+ / - , while the parent is Ctsk Cre + / - and Ctsk Cre- / - Under the premise, the F0 generation will not generate Ctsk Cre+ / + Therefore, the F0 generation only needs to detect Ctsk Cre+ / - That is, in the F0 generation screening, as long as the primer pair of Ctsk gene is used for PCR amplification, the genotype of the amplified band is Ctsk Cre+ / - In the present invention, Rosa26iDTR preferably includes Rosa26iDTR flox / + and Rosa26iDTR flox / flox In the present invention, the parent Rosa26iDTR flox / flox The mice were homozygous and were crossed with wild-type mice. The offspring genotype was Rosa26iDTR flox / + Therefore, the F0 generation of mice does not need to be identified for the insertion of the Rosa26-iDTR gene.

[0049] In the examples of the present invention, in order to prove the reliability of the results, the Ctsk gene and Rosa26iDTR of the F0 generation were tested to determine whether they were homozygous, heterozygous, or wild type.

[0050] The PCR identification method of the present invention for detecting whether the Ctsk gene or the Ctsk-e(2A-Cre-Wpre-pA)1 gene is inserted comprises the following steps:

[0051] In the present invention, the screening method for whether the Ctsk gene or Ctsk-e (2A-Cre-Wpre-pA) 1 gene is inserted is preferably performed by PCR amplification using a primer pair designed for the Ctsk gene. In the present invention, when identifying whether the Ctsk gene is inserted, the primer pair designed for the wild-type gene preferably includes Ctsk-e (2A-Cre-Wpre-pA) 1-WT-442bp-F having a nucleotide sequence as shown in SEQ ID NO. 1 and Ctsk-e (2A-Cre-Wpre-pA) 1-WT-442bp-R having a nucleotide sequence as shown in SEQ ID NO. 2. In the present invention, the Ctsk-e (2A-Cre-Wpre-pA) 1-WT-442bp-F and Ctsk-e (2A-Cre-Wpre-pA) 1-WT-442bp-R are preferably referred to as the first primer pair. In the present invention, the primer pair designed for Ctsk preferably includes Ctsk-e(2A-Cre-Wpre-pA)1-403bp-F having a nucleotide sequence as shown in SEQ ID NO. 3 and Ctsk-e(2A-Cre-Wpre-pA)1-403bp-R having a nucleotide sequence as shown in SEQ ID NO. 4. In the present invention, Ctsk-e(2A-Cre-Wpre-pA)1-403bp-F and Ctsk-e(2A-Cre-Wpre-pA)1-403bp-R are preferably referred to as the second primer pair.

[0052] When identifying whether the Ctsk gene or the Ctsk-e (2A-Cre-Wpre-pA) 1 gene is inserted, if only the first primer pair can amplify a 442 bp band, it indicates that the mouse is a wild-type mouse and the exogenous Ctsk-e (2A-Cre-Wpre-pA) 1 gene is not inserted; if the first primer pair can amplify a 442 bp band and the second primer pair can amplify a 403 bp band, it indicates that the mouse is a heterozygous mouse, the exogenous Ctsk-e (2A-Cre-Wpre-pA) 1 gene is inserted into one chromosome and the exogenous Ctsk-e (2A-Cre-Wpre-pA) 1 gene is not inserted into one chromosome; if only the second primer pair can amplify a 403 bp band, it indicates that the mouse is a homozygous mouse, and the exogenous Ctsk-e (2A-Cre-Wpre-pA) 1 gene is inserted into both chromosomes.

[0053] The present invention provides a PCR identification method for determining whether the Rosa26iDTR gene is inserted, comprising the following steps:

[0054] In the present invention, the method for screening whether the Rosa26iDTR gene is inserted is preferably PCR amplification using a primer pair for the Rosa26iDTR gene. In the present invention, when identifying whether the Rosa26iDTR gene is inserted, the primer pair designed for the wild-type gene preferably includes Rosa26-iDTR-WT-487bp-F having a nucleotide sequence as shown in SEQ ID No. 5 and Rosa26-iDTR-WT-487bp-R having a nucleotide sequence as shown in SEQ ID No. 6. In the present invention, the Rosa26-iDTR-WT-487bp-F and Rosa26-iDTR-WT-487bp-R are preferably referred to as the third primer pair. In the present invention, the primer pair designed for the Rosa26iDTR gene preferably includes Rosa26-iDTR-878bp-F having a nucleotide sequence as shown in SEQ ID No. 7 and Rosa26-iDTR-878bp-R having a nucleotide sequence as shown in SEQ ID No. 8. In the present invention, Rosa26-iDTR-878bp-F and Rosa26-iDTR-878bp-R are preferably referred to as the fourth primer pair.

[0055] The present invention identifies whether the Rosa26iDTR gene is inserted. If only the third primer pair can amplify a 487bp band, it indicates that the mouse is a wild-type mouse and the exogenous Rosa26iDTR gene is not inserted; if the third primer pair can amplify a 487bp band and the fourth primer pair can amplify an 878bp band, it indicates that the mouse is a heterozygous mouse, with the exogenous Rosa26iDTR gene inserted in one chromosome and the exogenous Rosa26iDTR gene not inserted in one chromosome; if only the fourth primer pair can amplify an 878bp band, it indicates that the mouse is a homozygous mouse, with the exogenous Rosa26iDTR gene inserted in both chromosomes.

[0056] The genotype of F0 mice was identified as Ctsk Cre+ / - Rosa26iDTR flox / + After the mice, the present invention preferably further comprises the step of changing the F0 generation genotype to Ctsk Cre+ / - Rosa26iDTR flox / + Mice and Ctsk Cre+ / - Rosa26iDTR flox / + Mice were hybridized to obtain F1 generation; the F1 generation genotype was screened to be Ctsk Cre+ / - Rosa26iDTR flox / flox The mouse model is one in which osteoclasts express the diphtheria toxin receptor.

[0057] The present invention will Ctsk Cre+ / - Rosa26iDTR flox / +Mice and Ctsk Cre+ / - Rosa26iDTR flox / + The offspring of mice can theoretically produce 9 genotypes, namely Ctsk-e(2A-Cre-Wpre-pA)1 + / - / Rosa26-iDTR + / + Mouse, Ctsk-e(2A-Cre-Wpre-pA)1 + / - / Rosa26-iDTR + / - Mouse, Ctsk-e(2A-Cre-Wpre-pA)1 - / - / Rosa26-iDTR + / - Mouse, Ctsk-e(2A-Cre-Wpre-pA)1 - / - / Rosa26-iDTR + / + Mouse, Ctsk-e(2A-Cre-Wpre-pA)1 + / - / Rosa26-iDTR - / - Mouse, Ctsk-e(2A-Cre-Wpre-pA)1 - / - / Rosa26-iDTR - / - Mouse, Ctsk-e(2A-Cre-Wpre-pA)1 + / + / Rosa26-iDTR + / + Mouse, Ctsk-e(2A-Cre-Wpre-pA)1 + / + / Rosa26-iDTR + / - mice and Ctsk-e(2A-Cre-Wpre-pA)1 + / + / Rosa26-iDTR - / - In the embodiment of the present invention, the F0 generation genotype is Ctsk Cre+ / - Rosa26iDTR flox / + Mice and Ctsk Cre+ / - Rosa26iDTR flox / + After hybridization of mice, no Ctsk was obtained Cre+ / + The homozygous mice, that is, the F1 generation in the embodiment of the present invention, only produce 6 genotypes.

[0058] The present invention preferably screens F1 generation target mice by PCR amplification, i.e., screens F1 generation mice for a mouse model expressing diphtheria toxin receptor in osteoclasts by PCR amplification. In the present invention, screening F1 generation mice preferably includes performing PCR amplification using primer pairs for the Rosa26iDTR and Ctsk genes; performing PCR amplification using the Rosa26iDTR primer pair, and the appearance of a band corresponding to the fourth primer pair; and performing PCR amplification using the Ctsk primer pair, and the appearance of a band corresponding to the second primer pair, thereby obtaining an animal model expressing diphtheria toxin receptor in osteoclasts.

[0059] In the present invention, Rosa26iDTR will be produced in the F1 generation - / - and Ctsk Cre- / - Since there are two genotypes, the F1 generation needs to be identified for both the Ctsk gene and the Rosa26iDTR gene. As long as the Rosa26iDTR and Ctsk genotypes are present in the target mice of the present invention, the F1 generation only needs to be able to amplify a band using the fourth primer pair for the Rosa26iDTR gene and a band using the second primer pair for the Ctsk gene. To better illustrate the specific genotype information of the F1 generation mice of the present invention, the present invention embodiment identified all possible genotypes of the mice using the above-mentioned method for identifying whether the Ctsk gene and Rosa26iDTR gene are inserted. The method for identifying whether the Ctsk gene and Rosa26iDTR gene are inserted has been described in detail above and will not be repeated here.

[0060] The animal model of osteoclasts expressing diphtheria toxin receptor constructed by the method described in the above technical solution of the present invention can specifically express diphtheria toxin receptor in osteoclasts, and then can specifically delete osteoclasts by injecting diphtheria toxin.

[0061] The present invention provides a method for constructing a transgenic animal model with osteoclast-specific deletion, comprising the following steps:

[0062] The animal model expressing diphtheria toxin receptor in osteoclasts constructed by the method described in the above technical solution is injected with diphtheria toxin to obtain a transgenic animal model with osteoclast-specific deletion.

[0063] The present invention does not specifically limit the injection method of diphtheria toxin; conventional injection methods in the art may be used. The present invention demonstrates, through examples, that the number of osteoclasts is significantly reduced after diphtheria toxin injection, further demonstrating that the osteoclast-specific deletion transgenic animal model constructed in the present invention achieves osteoclast-specific deletion.

[0064] The present invention provides an animal model in which osteoclasts express a diphtheria toxin receptor and / or a transgenic animal model with osteoclast-specific deletion for use in screening or developing drugs for treating bone tumors. The animal model in which osteoclasts express a diphtheria toxin receptor is obtained using the construction method described in the above technical solution or is constructed using the method described in the above technical solution; the transgenic animal model with osteoclast-specific deletion is constructed using the construction method described in the above technical solution. The animal model in which osteoclasts express a diphtheria toxin receptor and / or the transgenic animal model with osteoclast-specific deletion provided by the present invention can be used to screen drugs for treating bone tumors; the animal model in which osteoclasts express a diphtheria toxin receptor and / or the transgenic animal model with osteoclast-specific deletion provided by the present invention can be used to develop drugs for treating bone tumors.

[0065] The present invention provides an animal model in which osteoclasts express a diphtheria toxin receptor and / or a transgenic animal model with a specific osteoclast deletion for use in preparing a preparation for studying osteoclast function. The animal model in which osteoclasts express a diphtheria toxin receptor is obtained using the construction method described in the above technical solution or constructed using the method described in the above technical solution; the transgenic animal model with a specific osteoclast deletion is constructed using the construction method described in the above technical solution.

[0066] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1

[0068] A method for constructing a transgenic mouse model with osteoclast-specific deletion comprises the following steps:

[0069] 1. Source of experimental animals:

[0070] Ctsk-e(2A-Cre-Wpre-pA)1: purchased from Shanghai Model Organisms Technology Co., Ltd., the full name of the strain is C57BL / 6JSmoc-Ctsk em1(2A-Cre-Wpre-pA)Smoc , catalog number NM-KI-190019.

[0071] Basic information about the gene Ctsk-e(2A-Cre-Wpre-pA)1: NCBI ID 13038, MGI ID 107823. This gene marks osteoclasts. This mouse strain has a 2A-Cre-Wpre-polyA co-expression construct inserted before the 3'UTR of the endogenous mouse Ctsk gene. The genotype of Ctsk-e(2A-Cre-Wpre-pA)1 is Ctsk-e(2A-Cre-Wpre-pA)1. + / - mouse, referred to as CtskCre+ / - mouse.

[0072] Rosa26-iDTR: purchased from Suzhou Saiye Biotechnology Co., Ltd., the strain background is C57BL / 6J, the product number is C001477. The genotype of Rosa26-iDTR is Rosa26-iDTR + / + mice, also known as Rosa26iDTR flox / flox mouse.

[0073] Strain Description: Expression of the simian diphtheria toxin receptor (DTR) is blocked by an upstream loxP-Stop-loxP cassette.

[0074] 2. Specific steps:

[0075] 1. Ctsk-e(2A-Cre-Wpre-pA)1 transgenic mice and Rosa26-iDTR mice were housed separately in an SPF-rated animal facility. Selected healthy breeder mice with normal physique, bright fur color, and sexual maturity of 6-8 weeks of age. Specific criteria include: 1) Body physique: robust, well-proportioned, with strong limbs and no abnormalities such as head tilt. 2) Coat: Smooth, shiny fur that adheres closely to the body and is not easily shed, with no external injuries. 3) Eyes: Bright, lively eyes with no discharge from the corners of the eyes, and no abnormalities such as cataracts or blindness. 4) Respiration: Nostrils clean and breathing normal. 5) Feces: Anus clean and fecal pellets uniform. 6) Genitals: After selecting suitable breeder mice, observe whether male mice have genitalia missing, and female mice should be checked for double vaginas or vaginal closure. 7) Behavior: Quick reactions, natural and lively movements, a good appetite, and no abnormal behaviors such as circling or sluggishness. Ctsk-e(2A-Cre-Wpre-pA)1 transgenic mice and Rosa26-iDTR mice of different sexes were housed together in the evening, with a male-to-female mating ratio of 1:2. The next morning, the female mice were examined for the formation of a vaginal plug, indicating successful mating.

[0076] Successfully mated female mice were housed individually in cages with adequate feed and water, ensuring clean bedding, and regularly observed for their pre-partum status. F0 generation mice were born approximately 21 days after successful cohabitation. F0 generation pups were weaned 21 days after birth. A small ear tissue sample was obtained and DNA was extracted using a Selleck Rat Tail Direct PCR Kit for PCR analysis of the F0 generation mice's genotypes.

[0077] 2. When performing PCR to identify the mouse genotype, the primers used to identify whether the exogenous gene containing Ctsk-e(2A-Cre-Wpre-pA)1 was inserted are shown in Table 1; the primers used to identify whether the exogenous gene containing Rosa26-iDTR was inserted are shown in Table 2.

[0078] Table 1 Primers used to identify whether the exogenous gene containing Ctsk-e(2A-Cre-Wpre-pA)1 is inserted

[0079]

[0080] Table 2 Primers used to identify whether the exogenous gene containing Rosa26-iDTR is inserted

[0081]

[0082] When identifying whether an exogenous gene containing Ctsk-e(2A-Cre-Wpre-pA)1 has been inserted, the PCR identification procedure is as follows: 94°C initial denaturation for 5 minutes; 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 1 minute, 35 cycles; and 72°C final extension for 7 minutes. After obtaining the PCR product in the present invention, the PCR product can be temporarily stored at 4°C. When identifying whether the exogenous Rosa26-iDTR gene has been inserted, the PCR identification procedure is as follows: 94°C initial denaturation for 3 minutes; 94°C denaturation for 30 seconds, 62°C annealing for 35 seconds, 72°C extension for 35 seconds, 35 cycles; and 72°C final extension for 5 minutes. After obtaining the PCR product, the PCR product can be temporarily stored at 4°C.

[0083] The PCR system for the above PCR identification was: 0.5 μL each of F and R primers (10 μM), 10 μL of 2×M-PCR OPTITM Mix, 1 μL of DNA template, and H 2 O added to a total volume of 20 μL.

[0084] When the above-mentioned first primer pair and second primer pair are used to perform PCR on mouse DNA to identify whether the exogenous Ctsk-e (2A-Cre-Wpre-pA) 1 gene is inserted, if only the first primer pair can amplify a 442bp band, it means that the mouse is a wild-type mouse and the exogenous Ctsk-e (2A-Cre-Wpre-pA) 1 gene is not inserted; if the first primer pair can amplify a 442bp band and the second primer pair can amplify a 403bp band, it means that the mouse is a heterozygous mouse, with the exogenous Ctsk-e (2A-Cre-Wpre-pA) 1 gene inserted in one chromosome and not in the other chromosome; if only the second primer pair can amplify a 403bp band, it means that the mouse is a homozygous mouse, with the exogenous Ctsk-e (2A-Cre-Wpre-pA) 1 gene inserted in both chromosomes. Figure 1 The parental genotype shown is Ctsk-e(2A-Cre-Wpre-pA)1 + / - Mouse (Ctsk Cre+ / - Mouse) PCR identification results. Figure 1 HE in the parental genotype is Ctsk-e(2A-Cre-Wpre-pA)1 + / - Mouse (Ctsk Cre+ / - mice), WT refers to wild-type mice, the same below.

[0085] When the third primer pair and the fourth primer pair are used to perform PCR on mouse DNA to identify whether the exogenous Rosa26-iDTR gene is inserted, if only the third primer pair can amplify a 487bp band, it means that the mouse is a wild-type mouse and the exogenous Rosa26-iDTR gene is not inserted; if the third primer pair can amplify a 487bp band and the fourth primer pair can amplify an 878bp band, it means that the mouse is a heterozygous mouse, with the exogenous Rosa26-iDTR gene inserted in one chromosome and not in the other chromosome; if only the fourth primer pair can amplify an 878bp band, it means that the mouse is a homozygous mouse, with the exogenous Rosa26-iDTR gene inserted in both chromosomes. Figure 2 The parental genotype shown is Rosa26-iDTR + / + Mouse (Rosa26iDTR flox / flox Mouse) PCR identification results. Figure 2 The HO in the parental genotype is Rosa26-iDTR + / + mice, WT is wild-type mice.

[0086] The genotype of F0 generation mice was identified by the above-mentioned PCR identification method. + / - / Rosa26-iDTR + / - Mouse (ie, Ctsk Cre+ / - Rosa26iDTR flox / + The agarose gel electrophoresis results of mice Figure 3 shown. Figure 3 The numbers in the table are the numbers of mice, the same below.

[0087] Depend on Figure 3 It can be seen that mice 89, 90, 92, 93, 94 and 96 have the genotype Ctsk-e(2A-Cre-Wpre-pA)1 + / - / Rosa26-iDTR + / - Heterozygous mice.

[0088] 3. Screening F0 generation Ctsk-e(2A-Cre-Wpre-pA)1 + / - / Rosa26-iDTR + / - Double gene mice, F0 generation Ctsk-e (2A-Cre-Wpre-pA) 1+ / - / Rosa26-iDTR + / - Bigenic mice and Ctsk-e(2A-Cre-Wpre-pA)1 + / - / Rosa26-iDTR + / - The double gene mice were hybridized to obtain F1 generation mice, and the genotype of the F1 generation mice was identified using the PCR identification method in step 2 above. + / - / Rosa26-iDTR + / - mice and Ctsk-e(2A-Cre-Wpre-pA)1 + / - / Rosa26-iDTR + / + Mouse (Ctsk Cre+ / - Rosa26iDTR flox / + Mice and Ctsk Cre+ / - Rosa26iDTR flox / flox Agarose gel electrophoresis of mice Figure 4 shown.

[0089] Depend on Figure 4 It can be seen that the genotype of mice 52, 53 and 54 in the F1 generation is Ctsk-e(2A-Cre-Wpre-pA)1 + / - / Rosa26-iDTR + / - Mice; mice 60, 61, and 62 in the F1 generation were of the Ctsk-e(2A-Cre-Wpre-pA)1 genotype + / - / Rosa26-iDTR + / + mouse.

[0090] Ctsk-e(2A-Cre-Wpre-pA)1 in F0 and F1 generations + / - / Rosa26-iDTR + / - Mouse, Ctsk-e(2A-Cre-Wpre-pA)1 + / - / Rosa26-iDTR + / + The mouse is a mouse model in which diphtheria toxin receptor (DTR) targets osteoclasts, that is, a mouse model in which osteoclasts express diphtheria toxin receptor.

[0091] 4. The diphtheria toxin receptor (DTR)-targeted osteoclast mouse model was injected with diphtheria toxin to achieve osteoclast-specific deletion, thus obtaining a transgenic mouse model with osteoclast-specific deletion.

[0092] Example 2

[0093] Tartrate-Resistant Acid Phosphatase (TRAP) staining is a specific detection technique based on enzyme histochemistry. Its core principle relies on the unique biochemical properties of the TRAP enzyme and its high expression in osteoclasts. TRAP belongs to the acid phosphatase family, with an optimal catalytic pH of 4.5-5.5, and can release inorganic phosphate by hydrolyzing phosphate monoester bonds. Unlike other acid phosphatases (such as lysosomal acid phosphatases), TRAP has a characteristic biochemical property: it is highly resistant to the inhibitory effects of L(+)-tartaric acid. In conventional acid phosphatase reaction systems, tartaric acid (final concentration 50-100mM) can completely inhibit the activity of non-TRAP enzymes, while TRAP still retains approximately 80% of its catalytic ability. This selective inhibitory property lays the theoretical foundation for the specific labeling of osteoclasts. The enzymatic activity of TRAP is visualized using an azo-coupled colorimetric assay: 1) Substrate hydrolysis: Naphthol AS-BI phosphate, a synthetic substrate, is catalyzed by TRAP to produce naphthol AS-BI (6-bromo-2-hydroxy-3-naphthoic acid-2-methoxy anilide); 2) Azo dye formation: Free naphthol AS-BI undergoes a coupling reaction with a diazonium salt (such as Fast Red TR salt or Fast Red Violet LB salt) to produce an insoluble red / purple azo dye (λmax ≈ 500-520 nm), which precipitates at the enzyme active site, thereby localizing TRAP in tissues or cells. Osteoclasts, the primary functional cells for bone resorption, experience significant upregulation of TRAP expression during differentiation and maturation. This enzyme participates in bone resorption through two mechanisms: 1) Inorganic phase dissolution: In an acidic microenvironment (H+ pump secretion), TRAP may directly hydrolyze pyrophosphate on the surface of bone mineral, promoting the dissolution of hydroxyapatite; 2) Organic phase degradation: TRAP synergizes with cathepsin K to degrade organic components of the bone matrix, such as collagen. Therefore, a positive TRAP staining signal (red / purple granular deposits) not only marks the presence of osteoclasts but also indirectly reflects their bone resorption activity.

[0094] This study used a conditional gene knockout strategy to verify the osteoclast-specific clearance effect through a diphtheria toxin receptor (DTR)-mediated cell ablation system. The experimental animal model was Ctsk Cre+ / - Rosa26iDTR flox / flox Double transgenic mice in which the Ctsk promoter drives the expression of Cre recombinase specifically in osteoclast lineage cells, while the Rosa26 locus carries a loxP diphtheria toxin receptor (DTR) reporter gene.

[0095] The experimental design is as follows: 6-8 weeks old Ctsk Cre+ / -Rosa26iDTR flox / flox Mice were randomly divided into two groups: a control group (administered intraperitoneally with phosphate-buffered saline daily for 3 consecutive days) and an experimental group (administered intraperitoneally with 250 ng of diphtheria toxin (DT) daily for 3 consecutive days). Twenty-four hours after the last injection, mice were sacrificed, and bilateral femurs were dissected and fixed in 4% paraformaldehyde (4°C, 24 hours) and decalcified. Decalcified bone tissue was dehydrated with graded ethanol and embedded in paraffin, and 5-μm-thick coronal sections were prepared. Osteoclasts were detected using tartrate-resistant acid phosphatase (TRAP) histochemical staining. 1) Staining conditions: Sections were dewaxed with xylene and rehydrated with graded ethanol. The sections were then immersed in sodium acetate buffer (0.1 M, pH 5.0, containing 50 mM L(+)-tartaric acid) containing naphthol AS-BI phosphate (1.5 mg / mL) and Fast Red TR salt (0.3 mg / mL) and incubated at 37°C in the dark for 45 minutes.

[0096] Results Interpretation: Multinucleated (≥3 nuclei) and TRAP-positive osteoclasts (deposited dark red granules in the cytoplasm) were defined as mature osteoclasts. In the experimental group, the number of TRAP-positive, multinucleated cells, i.e., osteoclasts, was significantly reduced compared with the control group. Non-osteoclasts (such as osteoblasts and osteocytes) remained TRAP-negative, confirming that the ablation process was cell type-specific and that osteoclasts were specifically deleted.

[0097] Example 3

[0098] Inhibitory effect of osteoclast ablation on tumor growth in the MC38 bone tumor model

[0099] Ctsk constructed in Example 1 Cre+ / - Rosa26iDTR flox / flox Genetic mice were randomly divided into two groups, 6 in each group, the experimental group and the control group, to verify the specific elimination of osteoclasts by the diphtheria toxin receptor (DTR)-mediated cell ablation system. In the experimental design, each mouse was inoculated with MC38 cell suspension by subcutaneous injection (simulating primary lesions) and tibial plateau injection (simulating bone metastasis) to establish a bone tumor animal model. The subcutaneous injection volume was 5×10 5 cells / mouse, and the injection volume into the tibia was 2.5×10 5 The experimental group received intraperitoneal injection of diphtheria toxin (DT, 250 ng / time) 24 hours before tumor inoculation, and then maintained a DT administration schedule every 4 days; the control group received intervention with an equal volume of phosphate buffered saline (PBS).

[0100] Starting from the 10th day after inoculation, the tumor volume was dynamically monitored every 48 hours. The formula for measuring tumor volume is: V = 0.5 × L × W 2 (L: maximum diameter, W: minimum diameter in the vertical direction).

[0101] The tumor volume monitoring results of the experimental and control groups are shown in Table 3 and Figure 6 shown.

[0102] Table 3 Tumor volume monitoring results of the experimental group and the control group

[0103]

[0104]

[0105] Table 3 and Figure 6 Results showed that the continuous osteoclast depletion group exhibited a significant tumor growth inhibition effect compared to the control group (unpaired t-test, *p<0.05). Furthermore, osteoclast ablation not only effectively slowed tumor progression but also reduced tumor volume.

[0106] Example 4

[0107] Inhibitory effect of osteoclast ablation on tumor growth in the B16 bone tumor model

[0108] Ctsk constructed in Example 1 Cre+ / - Rosa26iDTR flox / flox Genetic mice were randomly divided into two groups, 5 in each group, the experimental group and the control group, to verify the specific elimination of osteoclasts through the diphtheria toxin receptor (DTR)-mediated cell ablation system. In the experimental design, each mouse was inoculated with B16 cell suspension through two routes of subcutaneous injection (simulating primary lesions) and tibial plateau injection (simulating bone metastasis) to establish a bone tumor animal model. The subcutaneous injection volume was 1×10 6 cells / mouse, and the injection volume into the tibia was 5×10 5 The experimental group received intraperitoneal injection of diphtheria toxin (DT, 250 ng / time) 24 hours before tumor inoculation, and then maintained a DT administration schedule every 4 days; the control group received intervention with an equal volume of phosphate buffered saline (PBS).

[0109] Starting from the 10th day after inoculation, the tumor volume was dynamically monitored every 48 hours. The formula for measuring tumor volume is: V = 0.5 × L × W 2 (L: maximum diameter, unit: mm, W: minimum diameter in the vertical direction, unit: mm).

[0110] The tumor volume monitoring results of the experimental and control groups are shown in Table 4 and Figure 7 shown.

[0111] Table 4 Tumor volume monitoring results of the experimental group and the control group

[0112]

[0113]

[0114] Table 4 and Figure 7 Results showed that the continuous osteoclast depletion group exhibited a significant tumor growth inhibition effect compared to the control group (unpaired t-test, *p<0.05). Furthermore, osteoclast ablation not only effectively slowed tumor progression but also reduced tumor volume.

[0115] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for constructing an animal model in which osteoclasts express diphtheria toxin receptor, characterized in that: The following steps are involved: The gene expressing the simian diphtheria toxin receptor is inserted into osteoclasts, so that the gene of the simian diphtheria toxin receptor is specifically expressed in osteoclasts, thereby obtaining an animal model in which osteoclasts express the diphtheria toxin receptor.

2. The construction method according to claim 1, characterized in that: The species of animal include mammals that are resistant to diphtheria toxin.

3. A method for constructing an animal model of osteoclasts expressing diphtheria toxin receptor using the Cre-loxP method, characterized in that: The following steps are involved: The constructed Rosa26iDTR flox / flox Animals and Carrying Ctsk Cre+ / - Crossing the tool animal with the parent to obtain the F0 generation; the Cre gene carried by the tool animal is inserted before the 3'UTR of the Ctsk gene; Screening F0 generation genotype is Ctsk Cre+ / - Rosa26iDTR flox / + The animal model is one in which osteoclasts express diphtheria toxin receptor.

4. The method according to claim 3, characterized in that The genotype obtained by screening was Ctsk Cre+ / - Rosa26iDTR flox / + The F0 generation of animals also includes: The genotype is Ctsk Cre+ / - Rosa26iDTR flox / + Animals with genotype Ctsk Cre+ / - Rosa26iDTR flox / + The animals were hybridized to obtain the F1 generation; the F1 generation genotype was screened to be Ctsk Cre+ / - Rosa26iDTR flox / + and Ctsk Cre+ / - Rosa26iDTR flox / flox The animal model is one in which osteoclasts express diphtheria toxin receptor.

5. The method according to claim 3 or 4, characterized in that: F0 generation screening and / or F1 generation screening include performing PCR amplification; PCR amplification was performed using the primer pair with the nucleotide sequence Rosa26-iDTR-878bp-F shown in SEQ ID NO.7 and the nucleotide sequence Rosa26-iDTR-878bp-R shown in SEQ ID NO.8, and a band appeared; and PCR amplification was performed using the primer pair with the nucleotide sequence Ctsk-e(2A-Cre-Wpre-pA)1-403bp-F shown in SEQ ID NO.3 and the nucleotide sequence Ctsk-e(2A-Cre-Wpre-pA)1-403bp-R shown in SEQ ID NO.4, and a band appeared, thereby obtaining an animal model in which osteoclasts express diphtheria toxin receptor.

6. The method according to claim 5, characterized in that The primer pair used for screening also includes Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-F with a nucleotide sequence as shown in SEQ ID NO.1 and Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-R with a nucleotide sequence as shown in SEQ ID NO.2; The nucleotide sequence of Rosa26-iDTR-WT-487bp-F is shown in SEQ ID No. 5, and the nucleotide sequence of Rosa26-iDTR-WT-487bp-R is shown in SEQ ID No.

6.

7. The method according to claim 6, characterized in that When identifying whether the Ctsk gene or Ctsk-e(2A-Cre-Wpre-pA)1 gene is inserted, if only Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-F and Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-R can amplify a 442bp band, it means that the mouse is a wild-type mouse and no exogenous Ctsk-e(2A-Cre-Wpre-pA)1 gene is inserted; if Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-F and Ctsk-e(2A-Cre-Wpre-pA)1-WT-442bp-R can amplify a 442bp band, Ctsk-e(2A-Cre-Wpre-pA)1 is inserted. -pA)1-403bp-F and Ctsk-e(2A-Cre-Wpre-pA)1-403bp-R can amplify a 403bp band, indicating that the mouse is a heterozygous mouse, with the exogenous Ctsk-e(2A-Cre-Wpre-pA)1 gene inserted into one chromosome and the exogenous Ctsk-e(2A-Cre-Wpre-pA)1 gene not inserted into one chromosome; if only Ctsk-e(2A-Cre-Wpre-pA)1-403bp-F and Ctsk-e(2A-Cre-Wpre-pA)1-403bp-R can amplify a 403bp band, indicating that the mouse is a homozygous mouse, with the exogenous Ctsk-e(2A-Cre-Wpre-pA)1 gene inserted into both chromosomes; In identifying whether the Rosa26iDTR gene is inserted, if only Rosa26-iDTR-WT-487bp-F and Rosa26-iDTR-WT-487bp-R can amplify a 487bp band, it indicates that the mouse is a wild-type mouse and no exogenous Rosa26iDTR gene is inserted; if Rosa26-iDTR-WT-487bp-F and Rosa26-iDTR-WT-487bp-R can amplify a 487bp band, Rosa26-iDTR-WT-487bp-R can amplify a 487bp band. -F and Rosa26-iDTR-878bp-R can amplify an 878bp band, indicating that the mouse is a heterozygous mouse, with the exogenous Rosa26iDTR gene inserted into one chromosome and the exogenous Rosa26iDTR gene not inserted into the other chromosome; if only Rosa26-iDTR-878bp-F and Rosa26-iDTR-878bp-R can amplify an 878bp band, it indicates that the mouse is a homozygous mouse, with the exogenous Rosa26iDTR gene inserted into both chromosomes.

8. A method for constructing a transgenic animal model with osteoclast-specific deletion, characterized in that: The following steps are involved: The animal model expressing diphtheria toxin receptor in osteoclasts constructed by the method according to any one of claims 3 to 7 is injected with diphtheria toxin to obtain a transgenic animal model with osteoclast-specific deletion.

9. Use of an animal model in which osteoclasts express a diphtheria toxin receptor and / or a transgenic animal model with osteoclast-specific deletion in screening or developing drugs for treating bone tumors, wherein the animal model in which osteoclasts express a diphtheria toxin receptor is obtained by the construction method described in claim 1 or 2 or is constructed by the method described in any one of claims 3 to 7; and the transgenic animal model with osteoclast-specific deletion is constructed by the construction method described in claim 8.

10. Use of an animal model in which osteoclasts express a diphtheria toxin receptor and / or a transgenic animal model with osteoclast-specific deletion in the preparation of a preparation for studying osteoclast function, wherein the animal model in which osteoclasts express a diphtheria toxin receptor is obtained using the construction method described in claim 1 or 2 or is constructed using the method described in any one of claims 3 to 7; and the transgenic animal model with osteoclast-specific deletion is constructed using the construction method described in claim 8.

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