Construction method and application of LAG3 gene humanized animal model

An animal model and humanized technology, applied in the field of animal genetic engineering and genetic modification, can solve problems such as impact consistency, difference in affinity between drug targets and receptors, failure, etc., to ensure success rate and increase homologous recombination The effect of repair chance

Active Publication Date: 2019-06-25
GEMPHARMATECH CO LTD
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  • Summary
  • 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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  • Construction method and application of LAG3 gene humanized animal model
  • Construction method and application of LAG3 gene humanized animal model
  • Construction method and application of LAG3 gene humanized animal model

Examples

Experimental program
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Embodiment 1

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

[0043] Use CRISPR Cas9 to replace Exon1, Exon2, Exon3, Exon4, Exon5, Exon6, and Exon7 of human LAG3 gene with Exon1, Exon2, Exon3, Exon4, Exon5, Exon6, Exon7 of mouse Lag3 gene, and establish LAG3 gene humanization kit Rat model. C57BL / 6 mouse is a relatively mature mouse strain at present, and C57BL / 6 mouse is a background mouse, and the LAG3 humanized mouse model has been successfully obtained.

[0044] 1. Determine the replacement area 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 retain mouse LAG3 The sequence of signal peptide, transmembrane region and intracellular region, and the selected human LAG3 gene sequence is shown in SEQ ID No.1.

[0046] SEQ ID No. 1

[0047]

[0048] 2. Screen...

Embodiment 2

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

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

[0079] Protein expression detection: F1 generation heterozygous mating can obtain homozygous, select C57BL / 6 background mouse, LAG3 humanized heterozygous and homozygous mouse spleen, grind and digest the tissue into single cells, use human anti-LAG3 antibody and mouse Source anti-LAG3 antibody, analyze the expression of humanized mouse protein by flow cytometry.

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

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

[0082] Digestion: 3ml of pre-cooled enzyme digestion solution (PBS containing Ca, Mg+2% CS+10mM HEPES+30ugDNase+1.75mg col...

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Abstract

The invention provides a method for preparing a LAG3 humanized animal model and application thereof. The method utilizes a CRISPR / Cas9 gene editing technology to replace a part of an extracellular region encoded by a mouse Lag3 gene with a corresponding human LAG3 gene fragment, simultaneously reserves an intracellular signal transduction region of the mouse Lag3 gene, and ensures that correct conduction of cell signals is not affected. The 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 specifically relates to a method for constructing a LAG3 genetically modified humanized animal model. Background technique [0002] Complex biological processes usually require in vivo analysis, and the research of human biology is severely restricted by morals and technology. Therefore, animal models are increasingly needed for in vivo research of human cells, tissues and organs. At present, scientists have developed a variety of humanized mouse or human-mouse chimeras to overcome these limitations, and they have now become important tools for in vivo research on human cells and tissues. [0003] In the 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 chemotherapeutic drugs...

Claims

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Application Information

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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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