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CN-121986755-A - Construction method of RUNX1 gene conditional knockout chronic myelogenous leukemia mouse model

CN121986755ACN 121986755 ACN121986755 ACN 121986755ACN-121986755-A

Abstract

The invention belongs to the technical field of mouse model construction, and particularly relates to a construction method of a RUNX1 gene conditional knockout chronic myelogenous leukemia mouse model. The method comprises the steps of hybridizing an RUNX1 gene conditional knockout mouse with an LYZ2-CreERT2 mouse to obtain an F1 generation, carrying out DNA identification and screening to obtain a double-transgenic mouse with a genotype LYZ2-CreERT2/RUNX1, hybridizing an F1 generation LYZ2-CreERT2/RUNX1 double-transgenic mouse with an SCL-tTA/Bcr-abl double-transgenic mouse to obtain an F2 generation, and carrying out DNA identification and screening to obtain a four-transgenic mouse with a genotype SCL-tTA/Bcr-abl/LYZ2-CreERT2/RUNX 1. The invention provides a solid research foundation for the deep research of the effect of RUNX1 genes in chronic myelogenous leukemia and the research and development of LSCs targeted drugs aiming at the RUNX1 genes.

Inventors

  • WANG WEIZHANG
  • CHEN JUNJIE
  • WU JIAXUAN
  • CHENG YUEYUN

Assignees

  • 广东药科大学

Dates

Publication Date
20260508
Application Date
20260212

Claims (9)

  1. 1. The method for constructing the RUNX1 conditional knockout chronic myelogenous leukemia mouse model is characterized by comprising the following steps of: s1, hybridizing a RUNX1 gene conditional knockout mouse with a LYZ2-CreERT2 mouse, and identifying to obtain an F1 generation mouse carrying LYZ2-CreERT2/RUNX1; S2, hybridizing an F1 generation LYZ2-CreERT2/RUNX1 double-transgenic mouse and an SCL-tTA/Bcr-abl double-transgenic mouse, and identifying to obtain the chronic myelogenous leukemia mouse model with the SCL-tTA/Bcr-abl/LYZ 2-CreERT2/RUNX1 genes carried by the F2 generation mouse, thus obtaining the RUNX1 gene conditional knockout.
  2. 2. The construction method according to claim 1, wherein in the steps S1 and S2, the F1 and F2 generation RUNX1 transgenic mice are identified by PCR method, the amplification reaction system is 25. Mu.L, wherein 12.5. Mu.L of PCR premix containing Tap DNA polymerase, 1.5. Mu.L of mouse DNA sample, 1. Mu.L of upstream primer (10. Mu.M), 1. Mu.L of downstream primer (10. Mu.M) and 9. Mu.L of ultrapure water are adopted, the reaction program of the PCR amplification system is 95 ℃ for 3min,95 ℃ for 15S,60 ℃ for 15S,72 ℃ for 5min, 5 steps for 35 cycles are repeated, 16 ℃ are adopted, the upstream primer sequence during RUNX1 genotype identification is shown in SEQ ID NO. 1, and the downstream primer sequence is shown in SEQ ID NO. 2.
  3. 3. The construction method according to claim 2, wherein the RUNX1 gene knockout result is judged by 1.2% agarose gel electrophoresis of wild-type mice having about 165bp bands, homozygous mice having about 233bp bands, and heterozygous mice having about 233bp and about 165bp bands.
  4. 4. The construction method according to claim 1, wherein in the steps S1 and S2, F1 and F2 generation LYZ2-CreERT2 transgenic mice are identified by PCR method, the amplification reaction system is 25. Mu.L, wherein the reaction conditions of the PCR amplification system are 94 ℃ 3min,94 ℃ 30S,58 ℃ 30S,72 ℃ 1min,72 ℃ 5min, 5 steps are repeated for 35 cycles, 16 ℃ are maintained, and the primers (10 pmol/. Mu.L) comprise 0.1. Mu.L of Tap DNA polymerase, 1. Mu.L of mouse DNA sample, 2. Mu.L of 10 xTaq PCR buffer, 2.5 mM dNTP 1. Mu.L, and the sequences of LYZ2-CreERT2 transgenic mice are shown as SEQ ID NO. 3-SEQ ID NO. 6.
  5. 5. The method according to claim 4, wherein the determination of LYZ2-CreERT2 gene knockout result is performed in 1.2% agarose gel electrophoresis of mice homozygous for the about 655bp band, mice heterozygous for the about 753bp band and about 655bp band, and wild-type mice for the about 753bp band and no about 655bp band.
  6. 6. The construction method according to claim 1, wherein in the step S2, the F2 generation Bcr-abl/SCL-tTA transgenic mice are identified by PCR method, the amplification reaction system is 20 μL, wherein the PCR containing Tap DNA polymerase is premixed by 10 μL, the sample of mouse DNA is 2 μL, the upstream primer (10 μM) is 1 μL, the downstream primer (10 μM) is 1 μL, the ultrapure water is 6 μL, the Bcr-abl/SCL-tTA transgenic mice are identified by the genotyping primer as shown in SEQ ID NO. 7-SEQ ID NO. 12, the reaction program of the PCR amplification system is 94 ℃ for 5min,94 ℃ for 30S,61 ℃ for 30S,72 ℃ for 1min,72 ℃ for 5min, and the steps are repeated for 34 cycles and 16 ℃ is maintained.
  7. 7. The construction method of claim 6, wherein the Bcr-abl gene identification result determination index is 1.2% agarose gel electrophoresis of about 550bp band, and an internal reference of about 324bp band is amplified, and wherein the SCL-tTA gene identification result determination index is 1.2% agarose gel electrophoresis of about 750bp band.
  8. 8. The construction method according to claim 1, wherein the constructed model of chronic myelogenous leukemia mice with conditional knockdown of the RUNX1 gene is applicable in LSCs targeted drugs against the RUNX1 gene.
  9. 9. A mouse model of chronic myelogenous leukemia with conditional knockdown of the RUNX1 gene constructed by the method of any one of claims 1-8.

Description

Construction method of RUNX1 gene conditional knockout chronic myelogenous leukemia mouse model Technical Field The invention belongs to the technical field of mouse model construction, and particularly relates to a construction method of a RUNX1 gene conditional knockout chronic myelogenous leukemia mouse model. Background The RUNX1 gene, acute myelogenous leukemia 1 (Acute myeloid leukemia 1, amL 1), was originally discovered from the chromosome 21 translocation breakpoint in the chromosome 21 translocation of t (8; 21) (q 22; q 22), and contained a 138 amino acid Runt homologous domain. RUNX1 belongs to the RUNX protein family, namely Runt related transcription factor (RUNX) protein family, which is a transcription factor family with highly conserved protein sequences, and regulates the expression of genes involved in cell differentiation and cell proliferation, and has important influence on many developmental and immunophysiologic processes, so that the loss of any RUNX family member adversely affects viability. Among them, RUNX1 is commonly found in acute myeloid leukemia (Acute myeloid leukemia), and plays an important role in the development of malignant diseases caused by various tumor cells due to its association with the growth and differentiation of blood tumor cells and solid tumor cells. Studies have shown that the RUNX1 gene plays a key role in regulating the development and precise maintenance of mammalian hematopoietic function, and is necessary for maintaining Hematopoietic Stem Cell (HSC) and progenitor cell homeostasis. Hematopoietic stem and progenitor cell expansion due to deletion of the RUNX1 gene may be an important cause of human leukemia. Researchers have also found that genes associated with RUNX1 target genes that regulate cell cycle and apoptosis may result in enhanced proliferation of cells with abnormal RUNX1 function. To study the function of RUNX1, literature and experimental models have employed Cre-LoxP conditional knockdown systems to knock down the RUNX1 gene at specific cell types or at specific developmental stages. For example, conditional knockdown of RUNX1 in myeloid cells was achieved using LysM-Cre or other myeloid-related Cre systems for studying its role in normal hematopoiesis or abnormal proliferation of myeloid lineage. However, the existing RUNX1 conditional knockout model is mainly used for basic hematopoietic function studies and is not systematically combined with a typical leukemia driving gene model. Chronic myeloid leukemia (chronic myeloid Leukemia, cmL) is a malignant clonal hematopoietic disease based on the Bcr-abl fusion gene. To study the mechanism of occurrence and means of drug intervention, various CmL mouse models have been established in the prior art, including animal models that utilize retrovirus or transgene to express the Bcr-abl fusion gene. Part of the literature reports that RUNX1 dysfunction may affect the progression of Bcr-abl related leukemias, e.g. RUNX1 mutations or dysfunctions are associated with disease progression in the acute phase. However, most of these studies are in vitro experiments or single genetic background analysis, or systematic integration using mutation/interference approaches, rather than conditional knockdown+classical CML transgene models. In the prior art, RUNX1 conditional knockdown models and Bcr-abl driven chronic myelogenous leukemia models are usually constructed and used independently of each other, and a stable genetic animal model for integrating RUNX1 functional deletions and Bcr-abl driven leukemia phenotypes at the body level is not yet available. The existing model is difficult to systematically evaluate the role of RUNX1 in the development, progression and progression of chronic myeloid leukemia. Therefore, there is a need to provide a new mouse model construction method to overcome the above-mentioned shortcomings. The construction of a chronic myelogenous leukemia mouse type of conditional knockout RUNX1 based on a chronic myelogenous leukemia mouse model (SCL-tTA/Bcr-abl mice) is discussed, and is important for defining the role of the RUNX1 gene in CmL diseases and the research and development of LSCs targeted drugs aiming at the RUNX1 gene. Disclosure of Invention The invention aims to provide a construction method of a RUNX1 gene conditional knockout chronic myelogenous leukemia mouse model. The technical scheme adopted by the invention for solving the technical problems is as follows: The invention relates to a construction method of a RUNX1 gene conditional knockout chronic myelogenous leukemia mouse model, which comprises the following steps: s1, hybridizing a RUNX1 gene conditional knockout mouse with a LYZ2-CreERT2 mouse, and identifying to obtain an F1 generation mouse carrying LYZ2-CreERT2/RUNX1; S2, hybridizing an F1 generation LYZ2-CreERT2/RUNX1 double-transgenic mouse and an SCL-tTA/Bcr-abl double-transgenic mouse, and identifying that the F2 generation mouse carries SCL-tTA/B