A method for constructing a mouse model of chronic myeloid leukemia with conditional overexpression of human EZH2

Abstract

The present invention belongs to the technical field of disease model construction, and particularly relates to a method for constructing a mouse model of chronic myeloid leukemia with conditional overexpression of human EZH2. The mouse model of chronic myeloid leukemia with conditional overexpression of human EZH2 constructed by the present invention finally obtains a mouse model in which the genes of Lyz2-creERT2, EZH2, BCR-ABL, and SCL-tTA are all positive and Lyz2-creERT2 and EZH2 are double homozygous through a series of hybridization and breeding processes, laying a solid research foundation for subsequent in-depth study of the role of histone methyltransferase EZH2 in the pathogenesis of chronic myeloid leukemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease model construction, and particularly relates to a method for constructing a mouse model of chronic myeloid leukemia with conditional overexpression of human EZH2. Background Art

[0002] Enhancer of Zeste Homolog 2 (EZH2) is a gene located on chromosome 7, which encodes histone methyltransferase EZH2, a core member of the polycomb group (PcG) family. The PcG family includes two polycomb complexes, PRC1 and PRC2, which can protect inactive genes from low-level or incorrect transcriptional activation signals and contribute to maintaining gene repression in many cell types. PRC2 contains four core components, EZH2 and its highly related homolog EZH1, suppressor of embryonic ectoderm development (EED), suppressor of zeste 12 (SUZ12), and retinoblastoma-associated proteins 46 and 48 (RbAp46 / 48). EZH2 is an important component of the PRC2 complex. There is a SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain located at the C-terminus in the functional domain of EZH2, and the transcriptional repression mediated by EZH2 protein depends on the intact SET domain. The methylation activity of EZH2 can methylate H3K27 mono-methyl, di-methyl, and tri-methyl, and H3K27 tri-methylation is considered to play a core role in the PcG gene silencing mechanism.

[0003] EZH2 is a gene essential for normal cells. Under normal physiological conditions, EZH2 can balance cell self-renewal and differentiation. In cells such as hematopoietic stem cells and osteoblasts, EZH2 can silence pro-differentiation genes, promote cell cycle progression, and prevent stem cell exhaustion. In addition, in other studies, it was found that EZH2 can participate in the methylation of non-histone substrates, such as participating in DNA methylation. By interacting with DNA methyltransferase, it promotes the binding of DNA methyltransferase to the promoter region of the target gene, thereby increasing the CpG methylation level. Under pathological conditions, in the study of tumors, mutations of EZH2 were found in a variety of malignant tumors, and the mutations occurred in the SET domain, enhancing its catalytic activity of H3K27 tri-methylation. Moreover, in the study of lymphoma diseases, it was found that compared with quiescent cells and proliferating lymphoma cells, the EZH2 gene was overexpressed in proliferating lymphoma cells, promoting the proliferation of cancer cells.

[0004] Previously, many studies have shown that the expression level of EZH2 in tissues such as gastric cancer and liver cancer is much higher than that in normal tissues. At the same time, in the research on myeloid malignancies, it was found that the EZH2 gene is usually overexpressed in myelodysplasia and acute myeloid leukemia, and overexpression of the EZH2 gene was also found in CML. Subsequently, the expression of the EZH2 gene was detected by PCR in the peripheral blood specimens of CML patients, and it was found that the expression of EZH2 was positively correlated with the occurrence of CML and was related to the activity of BCR-ABL1. It is speculated that BCR-ABL1 may positively regulate the expression of EZH2 through the STAT5 signaling pathway, resulting in simultaneous increases in the levels of EZH2 and phosphorylation of the transcription activator (STAT)5, and STAT5A is located in the promoter region of EZH2, leading to increased transcription of leukemia cells. To study this phenomenon, researchers applied an EZH2 inhibitor to the treatment of a chronic myeloid leukemia mouse model and achieved the expected therapeutic effect. These research reports all suggest that the expression of the EZH2 gene may be closely related to the occurrence and progression of chronic myeloid leukemia.

[0005] Therefore, constructing an animal model of chronic myeloid leukemia with overexpression of EZH2 is of great value for studying chronic myeloid leukemia. Summary of the Invention

[0006] In order to deeply study the role and mechanism of EZH2 in the pathogenesis of chronic myeloid leukemia, the present invention provides a human EZH2-overexpressing chronic myeloid leukemia mouse model and a method for constructing the same, which accelerates the research process of chronic myeloid leukemia.

[0007] The specific technical solution adopted by the present invention is as follows:

[0008] A method for constructing a human EZH2 conditionally overexpressing chronic myeloid leukemia mouse model, comprising the following process:

[0009] S1. Hybridize SCL-tTA mice under the FUV background with BCR-ABL mice, and identify and obtain F1 generation mice carrying the SCL-tTA / BCR-ABL gene;

[0010] S2. Hybridize the F1 generation mice carrying the SCL-tTA / BCR-ABL gene in step S1 with mice of the C57 background, screen out F2 offspring carrying the SCL-tTA / BCR-ABL gene and having brown hair, further breed, identify and screen to obtain C57 background F3 offspring carrying the SCL-tTA / BCR-ABL gene and having black hair;

[0011] S3. Hybridize EZH2 transgenic mice with LYZ2-CreERT2 transgenic mice and conduct identification to obtain C57 background mice with the F1 generation genotype of EZH2 / LYZ2-ERTCre2 heterozygosity;

[0012] S4. Breed the C57 background mice with the F1 generation genotype of EZH2 / LYZ2-CreERT2 heterozygosity obtained in step S3, identify and screen to select F2 generation EZH2 / LYZ2-CreERT2 homozygous mice, and then breed the selected F2 generation mice to obtain F3 generation EZH2 / LYZ2-CreERT2 homozygous mice;

[0013] S5. Hybridize the F3 generation EZH2 / LYZ2-CreERT2 homozygous mice obtained in step S4 with the C57 background F3 offspring carrying the SCL-tTA / BCR-ABL gene and having black hair obtained in step S2 to obtain F4 generation C57 background EZH2 / LYZ2-CreERT2 / SCL-tTA / BCR-ABL mice, namely human EZH2 conditional overexpression chronic myeloid leukemia mice;

[0014] S6. Expand and breed the human EZH2 conditional overexpression chronic myeloid leukemia mice prepared in step S5, identify and screen to obtain F5 generation SCL-tTA / BCR-ABL positive EZH2 / LYZ2-CreERT2 homozygous individuals, and that's it.

[0015] Preferably, in steps S1 - S2 and S5 - S6, the identification of SCL-tTA and BCR-ABL genotypes adopts the PCR method. The amplification system is 20 μL, including 2 μL of ddH2O, 4 μL of 5×Platinum TM GC Enhancer, 10 μL of 2×Platinum TM Direct PCRUniversal Master Mix, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), and 2 μL of DNA template.

[0016] Preferably, when identifying the SCL-tTA genotype, the upstream primer sequence is as shown in SEQ ID NO.1, and the downstream primer sequence is as shown in SEQ ID NO.2; when identifying the BCR-ABL genotype, the upstream primer sequence is as shown in SEQ ID NO.3, and the downstream primer sequence is as shown in SEQ ID NO.4.

[0017] Preferably, the reaction program of the PCR amplification system is as follows: 94°C for 5 min, 94°C for 30 s, 61°C for 30 s, 72°C for 1 min; repeat these three steps for 34 cycles; then 72°C for 5 min; 16°C for 20 min to obtain the product.

[0018] Preferably, the criterion for judging the BCR-ABL genotype identification result is the appearance of a 550-bp band and the internal reference of a 324-bp band amplified simultaneously; the criterion for judging the SCL-tTA genotype identification result is the appearance of a 750-bp band.

[0019] Preferably, in steps S3 to S6, the identification of the EZH2 and LYZ2-CreERT2 genotypes is carried out by PCR. The amplification system is 20 μL in total, including 10 μL of 2×Platinum TM Direct PCR UniversalMasterMix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 4 μL of 5×Platinum TM GCEnhancer, 3 μL of ddH2O, and 2 μL of DNA template.

[0020] Preferably, the sequence of upstream primer 1 for EZH2 genotype identification is as shown in SEQ ID NO.5, and the sequence of downstream primer 1 is as shown in SEQ ID NO.6; the sequence information of upstream primer 2 is as shown in SEQ ID NO.7, and the sequence information of downstream primer 2 is as shown in SEQ ID NO.8; the sequence of upstream primer 1 for LYZ2-CreERT2 genotype identification is as shown in SEQ ID NO.9, and the sequence of downstream primer 1 is as shown in SEQ ID NO.10; the sequence of upstream primer 2 is as shown in SEQ ID NO.11, and the sequence information of downstream primer 2 is as shown in SEQ ID NO.12.

[0021] Preferably, the reaction program of the PCR amplification system is as follows: 94°C for 2 min, 94°C for 15 s, 60°C for 15 s, 68°C for 20 s, repeat these three steps for 38 cycles; 4°C for 20 min to obtain the product.

[0022] Preferably, the criterion for judging the EZH2 gene identification result is as follows: only the appearance of a 994-bp band indicates wild type, the simultaneous appearance of 994-bp and 617-bp bands indicates heterozygous, and only the appearance of a band of approximately 617 bp indicates homozygous; the criterion for judging the LYZ2-CreERT2 gene identification result is as follows: only the appearance of a 753-bp band indicates wild type, the simultaneous appearance of bands of approximately 655 bp and 753 bp indicates heterozygous, and only the appearance of a band of approximately 655 bp indicates homozygous.

[0023] The present invention also provides a human EZH2 conditional overexpression chronic myeloid leukemia mouse model constructed by using the above method.

[0024] The object of the present invention is to construct a human EZH2 conditional overexpression chronic myeloid leukemia mouse model, that is, a transgenic mouse model of Lyz2-CreERT2 / EZH2 homozygous and having BCR-ABL and SCL-tTA. First, the genotypes of the mice were identified by PCR method. BCR-ABL expression was induced for 30 days after withdrawal of doxycycline, and after tamoxifen-induced EZH2 overexpression in the experimental group, the expression levels of EZH2 and BCR-ABL proteins in the spleen, kidney, liver and bone marrow cells of the mice were detected by western blotting method. The results showed that: the PCR results of the mice showed that offspring mice with positive Lyz2-creERT2, EZH2, Bcr-abl and SCL-tTA genes and homozygous Lyz2-creERT2 and EZH2 were obtained. The western blotting detection results showed that BCR-ABL protein was expressed in the bone marrow cells of LEB mice in the experimental group and the control group, and the expression level in the bone marrow cells of the experimental group of LEB mice induced by tamoxifen was significantly higher than that of the control group of LEB mice without tamoxifen induction, and BCR-ABL protein was not expressed in other tissues; ABL protein could be detected in the spleen tissues of LEB mice in the experimental group and the control group, and was not expressed in other tissue cells; EZH1 protein was expressed in the spleen, kidney and liver of LEB mice in the experimental group and the control group, and was not expressed in bone marrow cells, and the expression in the spleen tissue was significantly higher than that in other tissues; EZH2 was expressed in the bone marrow cells of the experimental group of LEB mice, and its expression could not be detected in other tissues of the experimental group or the control group of LEB mice, indicating that EZH2 was specifically induced to express in bone marrow cells.

[0025] Compared with the prior art, the technical advantages of the present invention are: the present invention has successfully constructed a human EZH2 overexpression chronic myeloid leukemia mouse model, laying a solid research foundation for the subsequent in-depth study of the role of histone methyltransferase EZH2 in the pathogenesis of chronic myeloid leukemia. Brief Description of the Drawings

[0026] Figure 1 It is a flowchart of the transgenic mouse hybridization process;

[0027] Figure 2 It is an identification result diagram of BCR-ABL and SCL-tTA genes of mouse No. B100 (SCL-tTA / Bcr-abl positive EZH2 / LYZ2-CreERT2 homozygous individual);

[0028] Figure 3Identification result diagram of the Lyz2-CreERT2 gene of mouse B100;

[0029] Figure 4 Identification result diagram of the EZH2 gene of mouse B100;

[0030] Figure 5 Spleen condition diagram of SCL-tTA / BCR-ABL mice 30 days after withdrawal of doxycycline induction of BCR-ABL expression;

[0031] Figure 6 Detection result diagram by westernbloting method. Specific implementation manners

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] The specific materials, instruments, etc. involved in the experiments of the present invention are as follows:

[0034] 1. Experimental materials:

[0035] The C57BL / 6JSmoc-Gt(ROSA)26Sorem1(CAG-LSL-EZH2-IRES-luciferase-2A-tdTomato)Smoc mice used in the present invention are heterozygous Lyz2-CreERT2 mice, 2 males and 2 females; the C57BL / 6JSmoc-Lyz2em2(CreERT2-IRES-EGFP)Smoc is heterozygous EZH2 mice, 2 males and 2 females; the C57BL / 6JSmoc-Lyz2em2(CreERT2-IRES-EGFP)Smoc is heterozygous EZH2 mice, 2 males and 2 females; the C57BL / 6JSmoc-Lyz2em2(CreERT2-IRES-EGFP)Smoc is heterozygous EZH2 mice, 2 males and 2 females; , 2 females, weight 18-22g, 4-8 weeks old; the above mice were purchased from Shanghai South Model Organisms Technology Co., Ltd., and the SPF-grade FVB.Cg-Tg (Tal1-tTA, i.e. SCL-tTA mice) 19Dgt male transgenic mice and FVB / N-Tg (tetO-BCR-ABL1, i.e. BCR-ABL mice) 2Dgt / J transgenic mice used in the present invention were purchased from the National Genetic Engineering Mouse Resource Bank of Nanjing University, with animal quality certificate number: SCXK (Su) 2010-0001. The above mice were all raised in the SPF-grade clean environment of the Experimental Animal Center of Guangdong Pharmaceutical University, with the temperature controlled at 18-22°C, the humidity at 50-70%, and automatic light control (12h light / 12h dark). The mouse cages, feed and bedding were sterilized, and the drinking water was sterilized at high temperature and high pressure. The feeding room was disinfected daily, and the bedding was changed twice a week to ensure that the feeding environment met the SPF-grade animal environment requirements.

[0036] 2. Instruments and reagents:

[0037] The clean bench SW-CJ-2F was purchased from Shanghai Boxun Medical Biological Instrument Co., Ltd.; the desktop high-speed centrifuge Centri-5810R was purchased from Eppendorf, Germany; the ultra-distilled water instrument PROG0002 was purchased from Millipore, USA; the multifunctional microplate reader workstation Flex station3 was purchased from Moleculardevices, USA; the electronic analytical balance BSA124S-CW was purchased from Sarorius, Germany; the palm centrifuge LX-200 was purchased from Haimen Qilin Medical Instrument Factory; the medical low-temperature preservation box DW-25L262 was purchased from Haier Group; the electrophoresis instrument PowerPacBasic and the gel imaging system Gel Doc XR+GelDocumentationSystem 1708195 were purchased from Bio-Rad, USA; the transfer tank VE-186 and Tannon developer 5200Multi were purchased from Shanghai Tianneng Technology Co., Ltd.; the horizontal shaker TY80s was purchased from Jintan Guowang Instrument Factory; the PCR instrument Gene The 9700 was purchased from AB Company in the United States; the dry heater D1100 was purchased from Labnet Company in the United States; the electrophoresis apparatus DYY-11 was purchased from Beijing Liuyi Instrument Factory.

[0038] The Platinum TM The DirectPCR Universal Master Mix PCR kit and the Thermo Scientific DreamTaq Hot Start Green PCR Master Mix (2×) PCR kit were both purchased from Thermo Fisher Scientific Company in the United States; the DL2000 DNA marker was purchased from TakaRa Company in Japan; the tamoxifen was purchased from Sigmaaldrich Company in the United States; the RIPA lysis buffer, PMSF, BCA protein concentration assay kit (P0012S), Tris-BASE (ultra pure), 1 mol / L Tris-HCl (pH 6.8), 1.5 mol / L Tris-HCl (pH 8.8), SDS-PAGE loading buffer, BSA, Fraction V, Tween-20, and glycine were all purchased from Beyotime Biotechnology Co., Ltd.; the Ezh2 (D2C9) Rabbit mAb, c-Abl Antibody, HSP70 (D69) Antibody, BCR-ABL (b2a2 Junction Specific) (L99H4) Mouse mAb, and Anti-biotin HRP-linked Antibody were all purchased from CELL SIGNALING TECHNOLOGY Company in the United States; the EZH1 Polyclonal antibody was purchased from Proteintech Company in the United States; the BCR-ABL and SCL-tTA gene PCR primers (100 uM) were synthesized by Shanghai Sangon Biological Engineering Co., Ltd.; the EZH2 and LYZ2-CRE-ERT2 gene PCR primers (100 uM) were synthesized by Thermo Fisher Scientific Company in the United States; the protein marker was purchased from Thermo Fisher Scientific Company in the United States; the non-fat milk powder was purchased from BD Biosciences Company in the United States; the SDS was purchased from Nanjing Jiancheng Bioengineering Co., Ltd.

[0039] Example 1 Construction method of a human EZH2 overexpressing chronic myeloid leukemia mouse model

[0040] The construction process of the human EZH2 overexpressing chronic myeloid leukemia mouse model of the present invention is specifically as follows Figure 1Shown as follows: First, cross the SCL-tTA mice with FUV background with BCR-ABL mice. To improve the breeding rate, the breeding cages in the experiment generally contain 1 male and 2 females. Subsequently, identification is carried out. The obtained F1 generation carries the SCL-tTA / BCR-ABL gene. Subsequently, cross the obtained F1 generation mice with FUV background carrying the SCL-tTA / BCR-ABL gene with mice with C57 background, and screen out the F2 offspring carrying the SCL-tTA / BCR-ABL gene and having brown hair. Then, carry out breeding and identification to screen out the F3 offspring with C57 background carrying the SCL-tTA / BCR-ABL gene and having black hair.

[0041] Then cross the EZH2 transgenic mice and LYZ2-CreERT2 transgenic mice and carry out identification. The obtained F1 generation is C57 background mice with the genotype of EZH2 / LYZ2-CreERT2 heterozygosity. Expand and breed the F1 generation EZH2 / LYZ2-CreERT2 heterozygous mice, identify and screen them to obtain a small number of F2 generation EZH2 / LYZ2-CreERT2 homozygous mice. Breed these F2 generation homozygous mice to obtain F3 generation EZH2 / LYZ2-CreERT2 homozygous mice. Cross the obtained F3 generation EZH2 / LYZ-CreERT2 homozygous mice with the above-mentioned F3 generation C57 background SCL-tTA / BCR-ABL mice to obtain F4 generation C57 background EZH2 / LYZ2-CreERT2 / SCL-tTA / BCR-ABL mice, that is, human EZH2 conditional overexpression chronic myeloid leukemia mice. Expand and breed the F4 generation mice, identify and screen them to obtain F5 generation SCL-tTA / BCR-ABL positive EZH2 / LYZ2-CreERT2 homozygous individuals and preserve the species.

[0042] Identification of mouse genotype: (1) Extraction of genomic DNA from mouse toes: Cut off the mouse toes and put them into a numbered 1.5 mL centrifuge tube and cover the lid. Add 20 μL of Lysis Buffer and 0.8 μL of proteinase K to each tube and tighten the lid. Place it at room temperature for 10 min and then place it in a 98 °C constant temperature metal bath for 1 min to obtain DNA, and store it in a 4 °C environment.

[0043] (2) PCR identification: The primer sequences for identifying the genotype of SCL-tTA transgenic mice are as follows: SCL-tTA-F: 5’-TTTCGATCTGGACATGTTGG-3’ (SEQ ID NO.1);

[0044] SCL-tTA-R: 5’-TGCTTACATTTCCCCCTTTG-3’ (SEQ ID NO.2);

[0045] The primer sequences for genotyping BCR-ABL transgenic mice are as follows: BCR-ABL-F: 5'-GAGCGTGCAGAGTGGAGGGAGAACA-3' (SEQ ID NO.3);

[0046] BCR-ABL-R: 5'-GGTACCAGGAGTGTTTCTCCAGACTG-3' (SEQ ID NO.4);

[0047] Internal reference:

[0048] BCR-ABL Control-F: 5'-CTAGGCCACAGAATTGAAAGATCT-3';

[0049] BCR-ABL Control-R: 5'-GTAGGTGGAAATTCTAGCATCATCC-3';

[0050] The primer sequences for genotyping EZH2 transgenic mice are as follows:

[0051] EZH2-F1: 5'-TCAGATTCTTTTATAGGGGACACA-3' (SEQ ID NO.5); EZH2-R1: 5'-TAAAGGCCACTCAATGCTCACTAA-3' (SEQ ID NO.6); EZH2-F2: 5'-GGTGTTGTCGGGGAAATCATCGTC-3' (SEQ ID NO.7); EZH2-R2: 5'-AGGAGCCTGCCAAGTAAC-3' (SEQ ID NO.8); The primer sequences for genotyping Lyz2-creERT2 transgenic mice are as follows:

[0052] Lyz2-creERT2-F1: 5'-TGAGGAAAAACAGAAGCCATTATT-3' (SEQ ID NO.9);

[0053] Lyz2-creERT2-R1: 5'-CCATCTCTTCTCAGCCCCCTTAC-3' (SEQ ID NO.10);

[0054] Lyz2-creERT2-F2: 5'-CTTTCGCTTTCCCCCTCCCTATTG-3' (SEQ ID NO.11);

[0055] Lyz2-creERT2-R2: 5’-ACTGCTCCCCTGTCCCTGTTCTGA-3’ (SEQ ID NO.12).

[0056] When identifying the SCL-tTA and BCR-ABL genotypes, the amplification system was 20 μL, including 2 μL of ddH2O, 4 μL of 5×Platinum TM GC Enhancer, 10 μL of 2×Platinum TM Direct PCR Universal Master Mix, 1 μL of the upstream primer (10 μM), 1 μL of the downstream primer (10 μM), and 2 μL of the DNA template.

[0057] The reaction program of the amplification system was as follows: 5 min at 94°C, 30 s at 94°C, 30 s at 61°C, and 1 min at 72°C; repeat these three steps 34 cycles; then 5 min at 72°C; 20 min at 16°C to obtain.

[0058] When identifying the EZH2 and LYZ2-CreERT2 genotypes, the total amplification system was 20 μL, including 10 μL of 2×Platinum TM Direct PCR Universal Master Mix, 0.5 μL of the upstream primer, 0.5 μL of the downstream primer, 4 μL of 5×Platinum TM GC Enhancer, 3 μL of ddH2O, and 2 μL of the DNA template.

[0059] The reaction program of the PCR amplification system was as follows: 2 min at 94°C, 15 s at 94°C, 15 s at 60°C, 20 s at 68°C, repeat these three steps 38 cycles; 20 min at 4°C to obtain.

[0060] (3) Agarose gel electrophoresis: Electrophorese each PCR product in step (2) on a 1.2% agarose gel, using EB as the nucleic acid dye, and perform electrophoresis for 20 min under the conditions of a voltage of 120 V and a current of 200 mA for detection.

[0061] BCR-ABL gene identification result determination criteria: Whether a band of approximately 550 bp appears, and at the same time, an internal reference band of approximately 324 bp is amplified;

[0062] SCL-tTA gene identification result determination criteria: Whether a band of approximately 750 bp appears;

[0063] Judgment criteria for Lyz2-creERT2 gene identification: If only a band of approximately 753 bp appears, it is wild-type; if bands of approximately 655 bp and 753 bp appear simultaneously, it is heterozygous; if only a band of approximately 655 bp appears, it is homozygous.

[0064] Judgment criteria for EZH2 gene identification: If only a band of approximately 994 bp appears, it is wild-type; if bands of approximately 994 bp and 617 bp appear simultaneously, it is heterozygous; if only a band of approximately 617 bp appears, it is homozygous.

[0065] Identification result: Finally, offspring (i.e., F4 generation) mice that could simultaneously detect positive for Lyz2-cre-ERT2, EZH2, BCR-ABL, and SCL-tTA genes and were double homozygous for Lyz2-creERT2 and EZH2 were obtained.

[0066] (4) Breeding F4 generation mice to obtain F5 generation mice: There were two breeding cages for F4 generation mice, and their genotypes were all positive for BCR-ABL / SCL-tTA and carried Lyz2-cre-ERT2 and EZH2 genes. They were breeding cage 1: B2♂B9♀B22♀, breeding cage 2: B16♂B23♀B30♀ (the mice were numbered in the order of B1 - B100 according to their birth order and ear-tagged before identification, a small amount of tissue was taken for identification, and mice that did not meet the standards were eliminated, only leaving mice that met the standards). When the F5 generation mice born in these breeding cages grew to 4 weeks old, they were identified and screened. The identification results of mice that met the standards of being positive for Lyz2-cre-ERT2, EZH2, BCR-ABL, and SCL-tTA genes and being double homozygous for Lyz2-creERT2 and EZH2 are shown in Table 1.

[0067] Table 1 Mice meeting the requirements in the identification results

[0068]

[0069] As can be seen from the above table, there were 10 mice in the F5 generation that were positive for Lyz2-creERT2 (LYZ), EZH2, BCR-ABL, and SCL-tTA (BT) genes. Among them, combining attachments Figure 2 , 3, 4, it can be found that the mouse numbered B100 simultaneously met the following in the BCR-ABL gene identification result: a band of approximately 550 bp appeared, and an internal reference band of approximately 324 bp was amplified simultaneously; in the SCL-tTA gene identification result: a band of approximately 750 bp appeared; in the Lyz2-creERT2 gene identification result: only a band of approximately 655 bp appeared; in the EZH2 gene identification result: only a band of approximately 617 bp appeared.

[0070] In summary, the present invention successfully constructed mice that are positive for Lyz2-creERT2, EZH2, BCR-ABL, and SCL-tTA genes and are double homozygous for Lyz2-cre-ERT2 and EZH2.

[0071] Example 2 Inducing the Expression of Target Genes

[0072] The mice that were positive for BCR-ABL and SCL-tTA genes and were double homozygous for Lyz2-creERT2 and EZH2 in the F5 generation finally screened in Example 1 were used for seed preservation and expansion. Some other mice that were heterozygous (either one of the Lyz2 gene and the EZH2 gene was heterozygous or both were heterozygous) and had Lyz2-creERT2, EZH2, BCR-ABL, and SCL-tTA genes were divided into an experimental group and a control group. The mice in the experimental group were injected with tamoxifen (the solvent was corn oil), and the control group was only injected with corn oil as the solvent. Usually, doxycycline was added to the drinking water of the mice to inhibit the expression of the BCR-ABL gene through the SCL-tTA gene, enabling the mice to grow, develop, and reproduce normally without getting sick. The specific usage was to add 0.5 g of doxycycline to 50 mL of water, and then take 1 mL from it and add it to 500 mL of water for the mice to drink. Therefore, the mice could be induced to get sick by stopping adding doxycycline to the drinking water of the mice.

[0073] Twenty-one days after withdrawing doxycycline, 0.1 mL / only of tamoxifen (20 mg / mL, the solvent was corn oil) was intraperitoneally injected into the experimental group LEB mice, and 0.1 mL / only of corn oil was intraperitoneally injected into the control group LEB mice. The drug was administered continuously for 5 days, once a day, and at a fixed time period. After the drug administration was completed, the mice were observed for 7 days first. If there was no abnormality, the next step was carried out.

[0074] Then, western blot was used to detect the protein expression. The specific process was as follows:

[0075] (1) One mouse from each of the experimental group and the control group was sacrificed by cervical dislocation and sampled.

[0076] (2) Extraction of liver, kidney, and spleen tissue proteins: Fresh tissue about the size of a soybean (about 50 mg) was cut with an ophthalmic scissors and placed in an EP tube. 200 μL of PBS was added to the EP tube, and it was ground with an electric grinding rod. After centrifugation at 3000 rpm for 10 min, the supernatant was discarded. The above steps were carried out on ice as much as possible. Then, 10 μL of PMSF was added dropwise to the EP tube. After adding 990 μL of RIPA lysis buffer, it was left to stand on ice for 20 min to fully lyse the tissue. The fully lysed tissue fluid was centrifuged at 4°C and 12000 g for 5 min; the supernatant was taken. The protein concentration of the sample was measured using a BCA protein concentration assay kit.

[0077] (3) Extraction of bone marrow cell proteins: Rinse the bone marrow cells of two femurs and tibias of mice with PBS into an EP tube, centrifuge at 3000 rpm for 10 min, discard the supernatant, add 5 μL of PMSF dropwise to the EP tube, add 495 μL of RIPA lysis buffer, and let it stand on ice for 25 min to fully lyse the cells. Centrifuge the fully lysed sample at 4°C and 12000 g for 5 min; take the supernatant. Measure the protein concentration of the sample using a BCA protein concentration assay kit.

[0078] (4) Detection results: The protein concentrations of each tissue cell and the final sample loading amounts are shown in Table 2 and Table 3 below:

[0079] Table 2 Detection results of the experimental group (tamoxifen-induced)

[0080]

[0081]

[0082] Table 3 Detection results of the control group (not induced with tamoxifen)

[0083]

[0084] As can be seen from Table 2 and Table 3 above, after calculating the concentration and total amount of the extracted proteins, considering that the total amount and concentration of the proteins extracted from the spleen are relatively low, the sample loading amount is 15 μg, and the sample loading amounts of the other tissues are 25 μg.

[0085] The specific western blot procedure is as follows:

[0086] (1) Preparation of SDS-PAGE gel: Wash the glass plates, sample combs, and baffles, rinse several times with double-distilled water, wipe with ethanol, and air dry. Place a baffle between the two washed glass plates, and after installation, pour 8% separating gel between the glass plates, immediately cover it with a layer of double-distilled water, and let it stand for about 20 min until the gel polymerizes. Pour off the upper layer of double-distilled water, blot dry with filter paper, pour 5% stacking gel, insert the sample comb, and let it stand for about 20 min. According to the volume of the electrophoresis tank, add an appropriate amount of 1× SDS-PAGE electrophoresis buffer to make the buffer cover the glass plates by more than 5 mm.

[0087] (2) SDS-PAGE gel electrophoresis for protein separation: After measuring the protein concentration of the sample, add an appropriate amount of SDS-PAGE loading buffer, place it in a 100°C metal bath and heat for 10 min, take the quantified protein for loading (refer to Table 2 and Table 3 for the sample loading amount and concentration, etc.). The sample is electrophoresed at a voltage of 75 V for 25 min. When the blue band enters the 8% separating gel, switch the voltage to 110 V and electrophorese for 90 min. When the blue band reaches the bottom of the SDS-PAGE gel, stop electrophoresis.

[0088] (3) Transfer membrane: Cut a suitable polyvinylidene fluoride (PVDF) membrane, soak it in methanol for 3 min, equilibrate for 2 min, and transfer it into 1× transfer buffer. Soak the filter paper and sponge for transfer membrane in 1× transfer buffer. Stack them in the order of "cathode - sponge pad - filter paper - gel - PVDF membrane - filter paper - sponge pad - anode", place them in the transfer tank, add 1× transfer buffer, and transfer the membrane at a voltage of 80 V under constant voltage for 2 h.

[0089] (4) Blocking: After the transfer of the membrane is completed, take out the PVDF membrane, wash the membrane 3 times with 1× washing buffer, and then add 1× blocking solution for blocking. Block it on a shaker at room temperature for 2 h.

[0090] (5) Incubate with antibodies: Add EZH2 antibody (1:1000), EZH1 antibody (1:1000), c-ABL antibody (1:1000), HSP70 (1:1000), BCR-ABL antibody (1:1000) diluted with 5% BSA dilution, and incubate overnight on a shaker at 4°C. After washing the membrane 3 times with 1× washing buffer, add secondary antibody HRP diluted with 5% BSA dilution and incubate on a shaker at room temperature for 1 h.

[0091] (6) Development: After washing the membrane with 1× western blot washing buffer, add ECL luminescent solution for color development, image with Tanon imaging system, and analyze the results.

[0092] (7) Test results: The WB test results are as shown in the appendix Figure 6 It can be seen that BCR-ABL protein is expressed in the bone marrow cells of LEB mice in both the experimental group and the control group, and the expression level in the bone marrow cells of LEB mice in the experimental group induced by tamoxifen is significantly higher than that in the control group of LEB mice without tamoxifen induction. BCR-ABL protein is not expressed in other tissues; ABL protein can be detected in the spleen tissues of LEB mice in both the experimental group and the control group, and no expression is seen in other tissue cells; EZH1 protein is expressed in the spleen, kidney and liver of LEB mice in both the experimental group and the control group, and no expression is seen in bone marrow cells, and the expression in spleen tissue is significantly higher than that in other tissues; EZH2 is expressed in the bone marrow cells of LEB mice in the experimental group, and its expression cannot be detected in other tissues of both the experimental group and the control LEB mice, indicating that EZH2 is specifically induced to express in bone marrow cells.

[0093] In summary, the present invention successfully constructs a transgenic mouse model of chronic myeloid leukemia with conditional overexpression of human EZH2, provides a new animal model for clarifying the mechanism of action of EZH2 in chronic myeloid leukemia, and lays a solid foundation for studying the role of EZH2 in chronic myeloid leukemia.

Claims

1. A method for constructing a mouse model of chronic myeloid leukemia with conditional overexpression of human EZH2, characterized in that, It includes the following processes: S1. Hybridize SCL-tTA mice with BCR-ABL mice, and identify and obtain F1 generation mice carrying the SCL-tTA / BCR-ABL gene; S2. Hybridize the F1 generation mice carrying the SCL-tTA / BCR-ABL gene in step S1 with C57-background mice, screen out F2 offspring carrying the SCL-tTA / BCR-ABL gene and having brown hair, further breed, identify and screen to obtain C57-background F3 offspring carrying the SCL-tTA / BCR-ABL gene and having black hair; S3. Hybridize EZH2 transgenic mice and LYZ2-CreERT2 transgenic mice and conduct identification to obtain C57-background F1 generation mice with the genotype of EZH2 / LYZ2-ERTCre2 heterozygosity; S4. Breed the C57-background F1 generation mice with the genotype of EZH2 / LYZ2-CreERT2 heterozygosity obtained in step S3, identify and screen to screen out F2 generation EZH2 / LYZ2-CreERT2 homozygous mice, and then breed the screened F2 generation mice to obtain F3 generation EZH2 / LYZ2-CreERT2 homozygous mice; S5. Hybridize the F3 generation EZH2 / LYZ2-CreERT2 homozygous mice obtained in step S4 with the C57-background F3 offspring carrying the SCL-tTA / BCR-ABL gene and having black hair obtained in step S2 to obtain F4 generation C57-background EZH2 / LYZ2-CreERT2 / SCL-tTA / BCR-ABL mice, namely human EZH2 conditional overexpression chronic myeloid leukemia mice; S6. Expand and breed the human EZH2 conditional overexpression chronic myeloid leukemia mice prepared in step S5, identify and screen to obtain F5 generation SCL-tTA / BCR-ABL positive EZH2 / LYZ2-CreERT2 homozygous individuals, and that's all.

2. The construction method according to claim 1, characterized in that In steps S1 to S2 and S5 to S6, the genotypes of SCL-tTA and BCR-ABL were identified by PCR. The amplification system was 20 μL, including 2 μL of ddH2O, 4 μL of 5×Platinum TM GC Enhancer, 10 μL of 2×Platinum TM Direct PCR Universal Master Mix, 1 μL of upstream primer, 1 μL of downstream primer, and 2 μL of DNA template.

3. The construction method according to claim 2, characterized in that, The upstream primer sequence for SCL-tTA genotype identification is shown as SEQ ID NO.1, and the downstream primer sequence is shown as SEQ ID NO.2; the upstream primer sequence for BCR-ABL genotype identification is shown as SEQ ID NO.3, and the downstream primer sequence is shown as SEQ ID NO.

4.

4. The construction method according to claim 2, wherein The reaction program of the amplification system is: 94°C for 5 min, 94°C for 30 s, 61°C for 30 s, 72°C for 1 min; repeat these three steps for 34 cycles; then 72°C for 5 min; 16°C for 20 min to obtain.

5. The construction method according to claim 2, characterized in that The judgment standard for the BCR-ABL genotype identification result is the appearance of a 550 bp band and at the same time the amplification of a 324 bp band as an internal reference; the judgment standard for the SCL-tTA genotype identification result is the appearance of a 750 bp band.

6. The construction method according to claim 1, wherein In steps S3 - S6, the genotypes of EZH2 and LYZ2-CreERT2 were identified by PCR. The amplification system was a total of 20 μL, including 10 μL of 2×Platinum TM Direct PCRUniversalMasterMix, 0.5 μL of the upstream primer, 0.5 μL of the downstream primer, 4 μL of 5×Platinum TM GCEnhancer, 3 μL of ddH2O, and 2 μL of the DNA template.

7. The construction method according to claim 6, characterized in that, The sequence of upstream primer 1 for EZH2 genotype identification is shown as SEQ ID NO.5, and the sequence of downstream primer 1 is shown as SEQ ID NO.6; the sequence information of upstream primer 2 is shown as SEQ ID NO.7, and the sequence information of downstream primer 2 is shown as SEQ ID NO.8; the sequence of upstream primer 1 for LYZ2-CreERT2 genotype identification is shown as SEQ ID NO.9, and the sequence of downstream primer 1 is shown as SEQ ID NO.10; the sequence of upstream primer 2 is shown as SEQ ID NO.11, and the sequence information of downstream primer 2 is shown as SEQ ID NO.

12.

8. The construction method according to claim 6, wherein, The reaction program of the amplification system is as follows: 94°C for 2 min, 94°C for 15 s, 60°C for 15 s, 68°C for 20 s, repeat these three steps for 38 cycles; 4°C for 20 min, and that's it.

9. The construction method according to claim 6, characterized in that, The criteria for judging the results of EZH2 gene identification are as follows: if only the band of 994 bp appears, it is wild type; if both the bands of 994 bp and 617 bp appear, it is heterozygous; if only the band of 617 bp appears, it is homozygous; the criteria for judging the results of LYZ2-CreERT2 gene identification are as follows: if only the band of 753 bp appears, it is wild type; if both the bands of 655 bp and 753 bp appear, it is heterozygous; if only the band of 655 bp appears, it is homozygous.

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