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Construction method of CD3E gene modified humanized animal model

A construction method and gene modification technology, applied in chemical instruments and methods, biochemical equipment and methods, genetic engineering, etc., to achieve the effect of improving the immune system, ensuring the success rate, and overcoming the obstacles of immune reconstruction

Active Publication Date: 2019-02-19
GEMPHARMATECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no mouse model suitable for CD3E-TCB screening and evaluation on the market, which fills the gap in the market

Method used

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  • Construction method of CD3E gene modified humanized animal model
  • Construction method of CD3E gene modified humanized animal model
  • Construction method of CD3E gene modified humanized animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: Establishment of hCD3E mouse model

[0070] 1. Establishment and evaluation of hCD3E mouse model

[0071] We chose the BAC transgenic way to insert the human CD3E gene and its complete regulatory sequence into the mouse genome to establish the hCD3E transgenic mouse model. BALB / c mice are the most commonly used mouse strains in the field of tumor and immune research, and there are many syngeneic tumor cell lines available. Therefore, we used BALB / c as the background mouse to construct the hCD3E transgenic mouse model and successfully obtained two stable genetic lines. Both lines of mice can realize human CD3E (hCD3E) and mouse CD3e (mCD3e ) co-expression.

[0072] 2. Scheme design

[0073] The BAC containing the human CD3E gene is modified to remove other genes about 40kb away from CD3E, retaining the regulatory region of the human CD3E gene, and ensuring that the expression of human CD3E is controlled by the human promoter and regulatory sequences. fi...

Embodiment 2

[0117] Example 2 Verification of hCD3E expression in hCD3E BAC TG mice

[0118] We used flow cytometry (FACS) to detect the expression of mCD3e and hCD3E proteins in peripheral blood of different lines of hCD3E transgenic mice.

[0119] Method: 100ul of mouse peripheral blood was collected from the orbit (pre-added with EDTA.2K for anticoagulation), added 1×RBC, mixed well, room temperature protected from light, and the cell pellet was collected by centrifugation. After washing with FACS buffer, Fc block (CD16 / 32 antibody) antibody was incubated. After blocking, mCD3 antibody (biolegend, 100314) and hCD3 antibody (biolegend, 317304) were added into groups respectively. After incubation, wash twice with FACS buffer and detect with flow cytometry. Add Sytoxblue before loading on the machine to distinguish dead cells from live cells. The distribution ratio of mCD3e and hCD3E positive cell populations in lymphocytes was analyzed by FACS to screen the transgenic mouse line on th...

Embodiment 3

[0126] Example 3 Evaluation of immune system indicators in hCD3E BAC TG mice

[0127] The hCD3E humanized mice obtained from the establishment of the line should have a sound immune system, and the expression of hCD3E protein will not cause the disorder of the mouse immune system (severe defect of T / NK cells, etc.), which is effective for drug evaluation of hCD3E targets Sex matters. The immune indicators (mainly T / B / NK cells) of the mice were detected by FACS, and compared with wild-type mice, the immune system indicators of the mice were comprehensively determined. A humanized mouse model that can express functional hCD3E and has a sound immune system will be an important preclinical evaluation tool for evaluating the efficacy of antibodies based on hCD3E targets.

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Abstract

The invention uses a BAC (bacterial artificial chromosome) transgenic technique on BALB / c background to insert humanized CD3E and its complete regulation sequence into mouse genome and establishes a hCD3E transgenic mouse model which is applicable to screening drugs for colorectal cancer, hepatic cancer, melanoma, breast cancer, renal cancer and lymphoma; the range of bispecific antibody screeningtypes is greatly widened. The steps are optimized; the preparation success rate of the hCD3E transgenic mouse model is greatly increased; stably genetic mouse line is successfully attained at present. The mouse line allows co-expression of humanized CD3E(hCD3E) and murine CD3e(mCD3e); the CD3E humanized mouse has complete immune system; data are provided for the research and development of CD3E-TCB clinical drugs at home and aboard; the market blank is filled.

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 CD3E genetically modified humanized animal model based on microinjection and transgenic technology and its application in biomedicine. Background technique [0002] Complex biological processes often require in vivo analysis, and many important research advances have used mice as models for studying various biological systems. As the study of human biology in vivo is severely constrained ethically and technically, animal models are increasingly required to perform in vivo studies of human cells, tissues, and organs without putting individuals at risk. 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027
CPCA01K67/0278A01K2207/15A01K2217/05A01K2227/105A01K2267/0331C07K14/7051C12N15/8509
Inventor 赵静琚存祥张明坤贾冬景侯欢欢高翔
Owner GEMPHARMATECH CO LTD
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