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A kind of construction method and application of sirpa humanized animal model

An animal model, humanized technology, applied in the field of animal genetic engineering and genetic modification, can solve the problem of incomplete testing of antibodies in humans, and achieve the effect of high success rate

Active Publication Date: 2022-03-18
GEMPHARMATECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to species differences, the immune checkpoint antibodies screened in mice are not fully tested in humans

Method used

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  • A kind of construction method and application of sirpa humanized animal model
  • A kind of construction method and application of sirpa humanized animal model
  • A kind of construction method and application of sirpa humanized animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Establishment of SIRPA humanized mouse model

[0037] Using CRISPR Cas9 to replace the mouse Sirpa gene with human SIRPA on C57BL / 6 background and BALB / c background mice, thereby constructing a mouse model that can express human SIRPA. And by mating with PD1 humanized mice with independent property rights, a dual-human mouse model that can express human SIRPA and PD1 was obtained.

[0038] 1. Determine the replacement region of the human fragment and the inserted human sequence

[0039] According to the structure and function of human SIRPA, we selected the extracellular region encoded by the human SIRPA gene to replace the extracellular region encoded by the mouse Sirpa gene, retained the intracellular region of the mouse, and selected the extracellular region encoded by the human SIRPA gene The amino acid sequence (Aa: 28-362) is shown in SEQ No.1.

[0040] GVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFP RVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRK...

Embodiment 2B6

[0074] Embodiment 2B6-hSIRPA and BALB / c-hPD1 / hSIRPA mice Human SIRPA expression and immune system verification

[0075] The expression of SIRPA homozygous mice was analyzed by flow cytometry and the immune cell populations were 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.

[0076] 1. Protein expression detection

[0077] The thymus or spleen, which mainly expresses SIRPA, was selected to detect the expression of SIRPA protein in the tissues.

[0078] SIRPA protein flow detection method:

[0079] a) Material collection: Spleen of humanized mouse and background mouse were cut out, weighed, and placed in a C-tube.

[0080] b) Digestion: Enzyme digestion solution (PBS containing Ca, Mg+2% CS+10mM HEPES+30ugDNase+1.75mg collagenase D) was used to digest spleen and thymus for 30min at 37°C. Add 300ul of 0.1M EDTA to the digested ...

Embodiment 3B6

[0094] Example 3B6-hSIRPA humanized mice evaluate the tumor efficacy verification of anti-SIRPA antibodies

[0095] Since the verification data at the in vitro level is not enough to reflect the actual situation of hSIRPA in humanized mice, the present invention designs experiments to collect the following in vivo experimental data: Tumor-bearing B6-hSIRPA humanized mice anti-hSIRPA antibody anti-tumor drug Responsiveness and anti-tumor pharmacodynamic reactivity of anti-hSIRPA antibody combined with anti-mPDL1 antibody.

[0096] Detection method: MC38-hCD47 (murine CD47 humanized MC38 tumor cell line) cell culture. Resuscitated cells: Take them out of the liquid nitrogen tank, thaw them quickly in a 37°C water bath, inoculate them in a 15cm dish and culture them. Subculture: MC38-hCD47 is an adherent cell, and usually needs to be subcultured every 2-3 days. Subcutaneous injection of MC38-hCD47 cells: collect the cells, centrifuge at 1000 rpm for 5 min, discard the supernata...

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Abstract

The invention provides a method for preparing a humanized animal model of SIRPA. The method uses gene editing technology to replace the extracellular region encoded by the mouse Sirpa gene with the extracellular region encoded by the human SIRPA gene, and evaluates the immune system index and tumor The drug efficacy test tested the model, and confirmed that the obtained mouse model was successfully constructed. This model will facilitate the screening and evaluation of SIRPA-targeted drugs.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, in particular, relates to a method for constructing a humanized animal model of SIRPA gene modification based on gene editing technology. Background technique [0002] Cancer immunotherapy is a treatment that uses the body's own immune system to attack cancer cells. The confrontation between the immune system and cancer cells is a dynamic process of a long-term game, both head-on and intertwined. The immune cells of a healthy body can discover and kill most cancerous cells, but under the induction of various innate or acquired factors, the immune system will lose its absolute advantage, and even be "rebelled" by cancer cells, becoming a key factor in the occurrence and development of cancer. a member. [0003] With many tumor-modulating antibodies and checkpoint inhibitors approved and even more drugs being evaluated for clinical use, antibody-based cancer ther...

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
CPCC12N15/8509C12N15/113C12N15/907C07K14/70503A01K67/0276A01K67/0278C12N2310/20A01K2207/15A01K2217/072A01K2217/075A01K2227/105A01K2267/0331
Inventor 琚存祥李松张明坤侯欢欢赵静
Owner GEMPHARMATECH CO LTD
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