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A kind of construction method and application of tigit humanized mouse model

A mouse model, humanized technology, applied in the field of animal genetic engineering and genetic modification to ensure the success rate

Active Publication Date: 2022-04-08
GEMPHARMATECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The key to a suitable humanized animal model lies in the strategy of gene modification. Some TIGIT humanized mouse models currently on the market are humanized only with the IgV domain in the extracellular region; The coding sequence and the mouse transmembrane and intracellular coding sequence together with polyA are inserted behind the mouse initiation codon ATG. This method also replaces the mouse extracellular region, but removes all the sequences that regulate gene expression, which inevitably exists in the original gene. expression deviation

Method used

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  • A kind of construction method and application of tigit humanized mouse model
  • A kind of construction method and application of tigit humanized mouse model
  • A kind of construction method and application of tigit humanized mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Establishment of C57BL / 6-TIGIT humanized mouse model

[0050] 1. Determine the replacement region of the human fragment and the inserted human sequence

[0051] According to the binding domain of human TIGIT and PVR protein, we selected Exon1, intron1, Exon2, intron2 and Exon3 (transcript NM_173799.3) of human TIGIT gene sequence (Gene ID: 201633) to replace Exon1 and intron1 of mouse TIGIT , Exon2, intron2 and Exon3 (transcript NM_001146325.1), the transmembrane region and intracellular region of mouse TIGIT are retained, and the replacement strategy is shown in the figure figure 2 shown. The chimeric sequence of the replaced protein is as follows, the underlined part is the human replacement region:

[0052] MRWCLLLIWAQGLRQAPLASG MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAIC NADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP LGGTMAAAVLGLICLMVTGVTVLARKKSIRMHSIESGLGRTEAEPQEWNLRSLSSPGSPVQTQTAPAGPCGEQAEDDYADPQEYFNVLSYRSLESFIAVSKT...

Embodiment 2

[0087] Example 2 Human TIGIT expression and immune system verification in B6-hTIGIT mice

[0088] The expression of TIGIT homozygous mice was analyzed by flow cytometry and the immune cell population was checked. After analysis, the mice that could successfully express the humanized gene and did not cause obvious abnormalities in the immune system were available for tumor drug efficacy experiments.

[0089] 1. Protein expression detection

[0090] Thymus or spleen was selected as the main expression tissue of TIGIT, and the expression of TIGIT protein in the tissue was detected.

[0091] TIGIT protein flow detection method:

[0092] a) Sampling: Spleens were cut from B6-hTIGIT homozygous mice and C57BL / 6 background mice (see mouse information list), weighed, and placed in C-tubes.

[0093] b) Digestion: Peripheral blood was protected from light at room temperature and washed once with FACS buffer; spleen and thymus were digested with enzyme solution (PBS containing Ca, Mg+2%...

Embodiment 3B6

[0112] Example 3B6-hPD1 / hTIGIT mouse expression and functional verification of human TIGIT and PD1

[0113] 1. Mating PD1 pure and humanized mice with TIGIT humanized mice to obtain PD1 heterozygous TIGIT heterozygous humanized mice, male mice and PD1 pure and humanized mice were subjected to IVF to obtain a large number of PD1 pure and TIGIT heterozygous humanized mice, when the PD1 pure and TIGIT heterozygous humanized mice reach the appropriate age, the siblings are mated to obtain PD1 and TIGIT double pure and humanized mice, identification scheme and identification results As follows, the obtained PD1 and TIGIT double pure and humanized mice are numbered 148, 155, 161, 168, 172, 177-180, 182, 194#.

[0114] PCR Primer Information

[0115]

[0116]

[0117] Figure 8 – Figure 11 The electrophoresis identification diagram during the preparation of B6-hPD1 / hTIGIT mice is shown, in which Figure 9 The identification results of TIGIT-KI-target 5-end and 3-end are sh...

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Abstract

The invention provides a method for preparing a TIGIT humanized mouse model. The method utilizes CRISPR / Cas9 technology to humanize the extracellular region of mouse TIGIT and the intron part of the gene sequence of the extracellular region. All sequences in the membrane region and intracellular region, including introns and UTRs, are still of mouse origin. This method retains the internal regulatory sequences of the gene and the complete intracellular signal transduction ability. The humanized The model realizes the original endogenous expression characteristics of TIGIT, satisfies the maximization of the human region (antibody binding domain), avoids missing effective antibodies, and can be used to screen and evaluate drugs targeting the human TIGIT gene. It is an ideal preclinical drug Test the model.

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 TIGIT gene modified humanized animal model based on gene editing technology. Background technique [0002] In recent years, tumor immunotherapy has developed rapidly and has become the fourth-generation conventional treatment for tumors. The idea of ​​tumor immunotherapy is mainly carried out from the two aspects of targeting tumor cells and activating immune cells. There are four main categories of therapies that activate immune cells: tumor vaccines, non-specific cytokine therapy, cell therapy, and immune checkpoint inhibitors. Among them, immune checkpoint inhibitors are the main force in the development of new drugs for tumor treatment, and immune checkpoint inhibitors including PD1, PDL1 and CTLA4 have shown significant anti-tumor efficacy. Although these immune checkpoints have achieved considerable...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/113C12N15/12C12N15/90A01K67/027
CPCA01K67/0276A01K67/0278C12N15/113C12N15/8509C12N15/907C07K14/47A01K2267/0331A01K2217/072A01K2217/075A01K2227/105A01K2207/15C12N2310/20C12N2310/10
Inventor 赵静琚存祥杨笑柳张明坤侯欢欢高翔
Owner GEMPHARMATECH CO LTD
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