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Leukemia mouse model based on gene co-transfection technology and preparation method thereof

A mouse model, leukemia technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., to achieve the effect of high similarity and high success rate

Active Publication Date: 2013-01-09
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Application Information

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Problems solved by technology

The K-ras gene is another important member of the Ras family, which has a high homology with the N-ras gene, and it has been reported that K-ras also exists in AML patients G12D Mutation, the proportion of this mutation is close to the former, but in the occurrence of AML, K-ras G12D The impact of mutations on disease development has not yet been reported

Method used

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  • Leukemia mouse model based on gene co-transfection technology and preparation method thereof
  • Leukemia mouse model based on gene co-transfection technology and preparation method thereof
  • Leukemia mouse model based on gene co-transfection technology and preparation method thereof

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Embodiment 1

[0041] A leukemia mouse model established by gene co-transfection technology and a preparation method thereof, comprising the following technical steps:

[0042] Construction of 1K-ras mutant and AML1-ETO fusion gene lentiviral vector

[0043] Construction of 1.1 K-ras mutant fusion gene lentiviral vector

[0044] The coding sequence of the K-ras gene (NM_004985) was obtained by searching Genbank, and at the same time, combined with literature review, the mutation site information was determined, and the point mutation primers were designed as follows using Primer 5 software

[0045]

[0046] Total RNA was extracted from the patient's tissue, and the cDNA obtained by reverse transcription was used as a template to clone the K-Ras (G12D) mutant gene by PCR. 30 cycles to amplify the K-Ras mutant gene. The amplified product was electrophoresed on a 1% agarose gel, and the Tiangen recovery kit was used to recover the gel by tapping, digest, and connect to the lentiviral vector ...

Embodiment 2

[0082] Example 2: Identification of the mouse leukemia model prepared by the present invention

[0083] 1. Bone marrow cell surface staining analysis:

[0084] Bone marrow cell typing and identification: After staining bone marrow cells with antibodies against c-KIT, Gr-1, and Mic-1, flow cytometry was used to analyze the proportion of positive cells in each group, so as to determine the degree of malignancy of AML. The results showed that GFP + / c -Kit + / Mac-1 - / Gr-1 - Immature primitive and naive cells, indicating that the model mouse has a remarkable characterization of the proportion of AML cell differentiation.

[0085] 2. Molecular level identification:

[0086] (1) Total RNA extraction:

[0087]Collection and processing of tissue samples: Take 50-100mg of tissue and put it into 1ml Trizol, use an electric homogenizer to homogenize, homogenize for 10 seconds, and pause for 10 seconds. Take the Trizol sample cracked above, put it in Vortex for 15 seconds, and let...

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Abstract

The invention relates to the technical field of biology, in particular to a leukemia mouse model based on a gene co-transfection technology and a preparation method thereof. The preparation method of the leukemia mouse model comprises the following steps: performing construction and packaging of K-ras mutants and AML1-ETO fusion gene lentivirus vectors; performing bone marrow cell separation and virus infection condition monitoring; implanting infection cells in a mouse to build a leukemia animal model; and performing model identification. The method of building the leukemia mouse model in a mode of utilizing caudal vein injection to lead in the manual site-directed mutagenesis K-ras mutants and AML1-ETO fusion gene lentivirus vectors is adopted initiatively. The leukemia mouse model is high in success rate, pathological characteristics are high in similarity to morbidity conditions of clinical leukemia, and the novel animal model can be provided for leukemia extramedullary infiltration mechanism research, leukemia medicine screening and gene and molecule target treatment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a ras mutant and an AML1-ETO fusion gene co-transfected bone marrow cell transplantation to establish a leukemia mouse model and a preparation method thereof. Background technique [0002] Acute myeloid leukemia (AML) is a hematological malignancy in which bone marrow hematopoietic precursor cells proliferate abnormally, and is a type of leukemia. It is characterized by a surge of immature white blood cells in the bone marrow, making it unable to make healthy blood cells. AML is the most common acute leukemia in adults, and its incidence increases with age. AML can be idiopathic or result from radiation exposure or exposure to cancer-causing chemicals. At present, the main treatment method is chemotherapy to kill malignantly proliferating cancer cells. In order to better provide new strategies for treatment, it is extremely urgent to establish an animal model as close as possible t...

Claims

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Application Information

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IPC IPC(8): C12N15/867A01K67/027
Inventor 崔淑芳汤球赵善民刘志学余琛琳孙伟蔡丽萍徐晨袁卫
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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