A kind of construction method of cd3e gene modification humanized animal model
A construction method and animal model technology, applied in chemical instruments and methods, biochemical equipment and methods, genetic engineering, etc., to overcome the obstacles of immune reconstruction, ensure the success rate, and achieve the effect of a sound autoimmune system
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Embodiment 1
[0068] Embodiment 1: Establishment of hCD3E mouse model
[0069] 1. Establishment and evaluation of hCD3E mouse model
[0070] 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.
[0071] 2. Scheme design
[0072] 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. f...
Embodiment 2
[0116] Example 2 Verification of hCD3E expression in hCD3E BAC TG mice
[0117] We used flow cytometry (FACS) to detect the expression of mCD3e and hCD3E proteins in peripheral blood of different lines of hCD3E transgenic mice.
[0118] 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
[0125] Example 3 Evaluation of immune system indicators in hCD3E BAC TG mice
[0126] 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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