Construction method and application of PHF6 <delta> and JAK3<M511I> double-mutation acute T lymphocytic leukemia mouse model

A JAK3M511I, lymphocyte technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem of unclear internal regulation mechanism of malignant transformation of T lymphocytes, and achieve the effect of short passage time

Active Publication Date: 2021-10-26
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the internal regulatory mechanism of malignant transformation of T lymphocytes is still unclear. Targeted drugs targeting the classic signaling pathway of T-ALL can only delay the process of T-ALL

Method used

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  • Construction method and application of PHF6 &lt;delta&gt; and JAK3&lt;M511I&gt; double-mutation acute T lymphocytic leukemia mouse model
  • Construction method and application of PHF6 &lt;delta&gt; and JAK3&lt;M511I&gt; double-mutation acute T lymphocytic leukemia mouse model
  • Construction method and application of PHF6 &lt;delta&gt; and JAK3&lt;M511I&gt; double-mutation acute T lymphocytic leukemia mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Build VCPhf6 fl / y and Mx1-Cre; Phf6 fl / y mouse

[0025] The Cre-lox recombination system includes two elements, Cre recombinase and Lox site: Cre recombinase can specifically recognize and catalyze the recombination of fragments between two Lox sites. The Lox site is a sequence of 8 core nucleotide fragments with a total length of 34 nucleotides. The orientation and position of the Lox sites determine the functional effects of the Cre enzyme, including knockout, inversion, or translocation of chromosomal DNA. The Cre recombinase gene and the Lox site do not exist in the genome of the mouse, so it is usually necessary to integrate the Cre gene and the Lox site into the mouse embryonic stem cells by transgenic means and breed corresponding transgenic mice.

[0026] Vav is a member of the Rho / Rac / CDC42 family. As a GTP / GDP conversion factor, Vav plays an important role in regulating G protein. At the same time, it is also a signal molecule necessary for lymphocyte antig...

Embodiment 2

[0034] Build VCPhf6 fl / y +JAK3 M511I mice and Phf6 WT+JAK3 M511I mouse

[0035] killphf6 lox / Y and Vav1-Cre;phf6 lox / Y The femur, tibia and ilium were removed from the mouse, and the muscle and fat tissue around the bone marrow were removed. PBE (phosphate buffered saline containing 2% fetal bovine serum) was used to draw out the bone marrow cells with a 1ml syringe, and then enriched with biotin magnetic beads. Lin of Set Mouse - Cells were plated in 24-well plates treated with recombinant human fibronectin fragments (Retronectin), pre-stimulated by adding cytokines mSCF, TPO and Flt3 for 8-12 hours, and further infected with JAK3 produced by 293T M511I After 48 hours, the retrovirus was observed using a fluorescence microscope, and the infected cells were collected to detect GFP by flow cytometry + cell ratio. Recipient mice were irradiated with a lethal dose (9Gy) to remove bone marrow cells, and irradiated twice with an interval of 4 hours each time. Four hours afte...

Embodiment 3

[0038] Determination of VCPhf6 fl / y +JAK3 M511I GFP in peripheral blood, bone marrow and various organs of mice + Cell ratio and differentiation

[0039] Blood cell count: collect 20 μL of peripheral blood from the tail vein into a heparin anticoagulant tube with 180 μL of diluent, and use a blood cell counting instrument to measure VC Phf6 fl / y +JAK3 M511I mice and Phf6WT+JAK3 M511I Mouse peripheral blood cells were analyzed. Mainly detect red blood cell count, hemoglobin level, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, white blood cell and platelet count.

[0040] Morphological and histological observation of bone marrow, spleen and peripheral blood of mice: Peripheral blood of mice was collected from the tail vein, blood smears were prepared, and after Wright-Giemsa staining, the morphology of blood cells was observed under a microscope. Test every four weeks. On the day when the mice were sacrificed, peripheral blood and bone ma...

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Abstract

The invention discloses a construction method and an application of a PHF6 <delta> and JAK3<M511I> double-mutation acute T lymphocytic leukemia mouse model. The preparation method comprises the following steps: utilizing a Vav1-Cre mouse to mate with a Phf6<fl/fl> mouse to obtain Vav1-Cre; a Phf6<fl/Y> (Phf6<-/Y>) male mouse; mating the Mx1-Cre mouse and the Phf6<fl/fl> to obtain Mx1-Cre; Phf6<fl/Y>male mouse; respectively enriching Lin-cells in bone marrow of two male mice, infecting retroviruses overexpressing GFP-JAK3M511I, and transplanting GFP + cells into receptor mice irradiated by a lethal dose through caudal vein injection, so as to construct two T-ALL leukemia mouse models. The model provided by the invention can be rapidly passed in mice, simulates the leukemia process of a T-ALL patient with PHF6 gene and JAK3 gene double mutation, and is used for targeted therapy drug screening and curative effect evaluation.

Description

technical field [0001] The invention relates to the technical field of animal model construction, in particular to a PHF6 △ and JAK3 M511I The method and application of double mutant acute lymphoblastic leukemia mouse model construction. Background technique [0002] Leukemia is a malignant clonal disease of hematopoietic stem cells, manifested in the malignant transformation and abnormal proliferation of hematopoietic stem cells, the ability to invade other tissues, and the significant reduction of normal cells in the bone marrow. Acute lymphoblastic leukemia (T-ALL), as a major type of leukemia, originates from T lymphocyte progenitor cells. Abnormally proliferating primitive lymphocytes not only accumulate in the bone marrow, but also infiltrate extramedullary tissues, such as meninges, lymph nodes, gonads, and liver. T-ALL has a high incidence in both children and adults, accounting for 10%–15% of childhood lymphocytic leukemia (ALL) and 25% of adult lymphocytic leuke...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/65C12N15/54A01K67/027A61K49/00
CPCC12N15/86C12N15/8509C12N15/65C12N9/12A01K67/0271A61K49/0008C12Y207/10002C12N2740/10043C12N2800/107A01K2207/12A01K2217/15A01K2227/105A01K2267/0331
Inventor 汪晓敏侯帅兵袁卫平袁胜男初雅婧
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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