A method for constructing a lag3 gene humanized animal model and its application

An animal model, humanized technology, applied in the field of animal genetic engineering and genetic modification, can solve the problems of drug target and receptor affinity difference, influence consistency, failure, etc., to increase the probability of homologous recombination repair, The effect of ensuring the success rate

Active Publication Date: 2021-02-19
GEMPHARMATECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, most of the candidate drugs that pass the preclinical evaluation will fail in the clinical phase I. The differences in physiology and pathology between mice and humans are important factors: differences in the immune system of humans and mice, and many pathogenic factors and drugs in humans. Species specificity between mice, differences in the affinity of drug targets and receptors between humans and mice, and differences between human-derived tumors and mouse-derived tumors will affect the consistency of preclinical and clinical data

Method used

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  • A method for constructing a lag3 gene humanized animal model and its application
  • A method for constructing a lag3 gene humanized animal model and its application
  • A method for constructing a lag3 gene humanized animal model and its application

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Embodiment 1

[0042] Example 1: Establishment of LAG3 humanized mouse model

[0043] Using CRISPR Cas9 to replace Exon1, Exon2, Exon3, Exon4, Exon5, Exon6, and Exon7 of the mouse Lag3 gene with Exon1, Exon2, Exon3, Exon4, Exon5, Exon6, and Exon7 of the human LAG3 gene, a small humanized LAG3 gene was established. mouse model. C57BL / 6 mouse is a relatively mature mouse strain in research at present. C57BL / 6 mouse was used as the background mouse, and the LAG3 humanized mouse model was successfully obtained.

[0044] 1. Determine the replacement region of the human fragment and the inserted human sequence

[0045]According to the functional domain of human LAG3 protein, select Exon1, Exon2, Exon3, Exon4, Exon5, Exon6, Exon7 of human LAG3 sequence to replace Exon1, Exon2, Exon3, Exon4, Exon5, Exon6, Exon7 of mouse Lag3, and keep mouse LAG3 Signal peptide, transmembrane region and intracellular region sequence, the selected human LAG3 gene sequence is shown in SEQ ID No.1.

[0046] SEQ ID No...

Embodiment 2

[0077] Example 2: Expression and functional verification of LAG3 humanized mouse model

[0078] 1. LAG3 expression and functional verification in LAG3 humanized mice

[0079] Protein expression detection: F1 generation heterozygous mating can obtain homozygous, select C57BL / 6 background mice, LAG3 humanized heterozygous and homozygous mouse spleens, grind and digest the tissues into single cells, use human anti-LAG3 antibody and mouse The source anti-LAG3 antibody was used to analyze the expression of humanized mouse protein by flow cytometry.

[0080] The method of flow cytometric detection of protein is as follows:

[0081] Materials: 4-week-old C57BL / 6 background heterozygous and homozygous LAG3 humanized mice were injected intraperitoneally with 7.5ug CD3e antibody. After 24 hours of stimulation, the spleen was taken for flow cytometry detection, weighed, and placed in a C-tube.

[0082] Digestion: There is 3ml of pre-cooled enzyme digestion solution (PBS containing Ca, ...

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Abstract

The present invention provides a method for preparing a LAG3 humanized animal model and its application. The method uses CRISPR / Cas9 gene editing technology to replace the part of the mouse Lag3 gene encoding the extracellular region with the corresponding human LAG3 gene fragment, and at the same time The intracellular signal transduction region of the mouse Lag3 gene is retained to ensure that the correct conduction of cell signals is not affected. This model is an ideal animal model for screening human LAG3 inhibitor drugs.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to a method for constructing a humanized animal model of LAG3 gene modification. Background technique [0002] Complex biological processes often require in vivo analysis, and the study of human in vivo biology is severely constrained by ethics and technology, so animal models are increasingly required for in vivo studies of human cells, tissues, and organs. Currently, scientists have developed a variety of humanized mice or human-mouse chimeras to overcome these limitations and have now become important tools for in vivo studies of human cells and tissues. [0003] During the research and development of clinical drugs, mice are widely used in the preclinical safety and effectiveness evaluation of candidate drugs, such as the in vivo effectiveness evaluation of new antiviral drugs, tumor immunotherapy and the development of new chemother...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/12C12N15/113A01K67/027
Inventor 高翔赵静琚存祥张明坤陶裴裴侯欢欢
Owner GEMPHARMATECH CO LTD
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