Preparation method and application of humanized CD3 genetically-modified animal model

A humanized, non-human animal technology, applied in the field of biomedicine, can solve the problems of cumbersome model building process, low accuracy, high cost, reduce drug development risks, speed up the research and development process, save time and cost. cost effect

Active Publication Date: 2019-06-21
BIOCYTOGEN JIANGSU CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The construction process of this type of model is cumbersome and expensive, and the targeting and specificity in specific target research are not strong, and the accuracy of the results of using this type of model to study drug efficacy is not high

Method used

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  • Preparation method and application of humanized CD3 genetically-modified animal model
  • Preparation method and application of humanized CD3 genetically-modified animal model
  • Preparation method and application of humanized CD3 genetically-modified animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0166] Embodiment 1 sequence design (partial replacement)

[0167] According to the purpose of the invention, designed figure 2 The targeting strategy for partial sequence replacement of the Cd3ε gene and the design of the targeting vector are shown. The targeting vector includes 5' homology arm (SEQ ID NO: 1), 3' homology arm (SEQ ID NO: 2) and human DNA fragment (SEQ ID NO: 3), and was constructed on the recombinant vector for positive cloning The screened resistance gene, such as the neomycin phosphotransferase coding sequence Neo, is equipped with two site-specific recombination systems arranged in the same direction on both sides of the resistance gene, such as the Frt recombination site (or conventional LoxP recombination system). Further, a gene encoding a negative selection marker, such as the gene encoding diphtheria toxin A subunit (DTA), was constructed downstream of the 3' homology arm of the recombinant vector. image 3 A schematic diagram of the humanized mou...

Embodiment 2

[0168] Example 2 Homologous Recombination Fragment Primer Design and PCR Amplification

[0169] Primers were designed to amplify 7 homologous recombination fragments (A1, A2-1, A2-2, A3, B, C1, C2). The primer sequences are shown in Table 1.

[0170] Table 1 Homologous recombination fragments and their corresponding lengths and primer sequences

[0171]

[0172] Using BAC as a template, 7 homologous recombination fragments were amplified using KOD-plus-polymerase. Among them, A1, A3, B, C1, and C2 selected the bacterial artificial chromosome (Bacterial Artificial Chromosome, BAC) (number: RP23-140I1, hereinafter referred to as "bacterium containing mouse BAC") containing the mouse Cd3ε genome as a template, and A2- 1. A2-2 selects the bacterial artificial chromosome containing the human CD3ε genome (number: RP11-414G21, hereinafter referred to as "bacteria containing human BAC") as a template. The specific PCR reaction system and reaction conditions are as follows:

[01...

Embodiment 3

[0178] Example 3 Construction of Homologous Recombination Targeting Vector

[0179] The targeting vector construction process is as follows:

[0180] 1. Ligate the fragment B obtained in Example 2 to the pBs-Neo vector by enzyme-cut ligation (BamHI / NotI) to obtain the pBs-Neo-B plasmid, and send it to the sequencing company for verification;

[0181] 2. After connecting the fragments A1 and A2-1, A2-2 and A3 obtained in Example 2 using overlap PCR (phusion enzyme) (see Table 4 and 5 for the reaction system and conditions), send them to the sequencing company for sequencing to confirm A1+A2 After the -1 and A2-2+A3 fragments are connected correctly, the fragments are digested and connected to the pBs-Neo-B plasmid (HindIII / SwaI / XhoI) to obtain pBs-Neo-(A1+A2-1+A2-2+ A3+B) plasmid;

[0182] 3. The pBs-Neo-(A1+A2-1+A2-2+A3+B) plasmid was electrotransformed into bacteria containing human BAC to obtain the pBs-Neo-(AB) plasmid containing the AB fragment; the AB fragment (see fi...

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Abstract

The invention relates to a humanized genetically-modified non-human animal, in particular a genetically-modified rodent, but in particular to a genetically-modified mouse, and particularly relates toa building method of an animal model to express humanized CD3 protein and application of the animal model in the field of biomedicine.

Description

technical field [0001] This application relates to the establishment method and application of a humanized genetically modified animal model, in particular, it relates to a construction method based on a humanized CD3 genetically modified animal model and its application in the field of biomedicine. Background technique [0002] Experimental animal disease models are indispensable research tools for the study of the etiology and pathogenesis of human diseases, the development of prevention technologies and the development of drugs. However, due to the differences in the physiological structure and metabolic system between animals and humans, traditional animal models cannot well reflect the real conditions of the human body. It is an urgent need for the biomedical industry to establish disease models in animals that are closer to the physiological characteristics of humans . [0003] With the continuous development and maturity of genetic engineering technology, the replace...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/90C12N9/22A01K67/027C07K19/00C12N15/62A61K49/00
CPCA01K67/0275A01K2207/15A01K2217/072A01K2217/077A01K2227/105A01K2267/0387A01K2267/0331C07K14/7051G01N33/5011G01N33/5088G01N33/56972A61K49/0008A01K67/0278A61K35/51
Inventor 沈月雷郭雅南黄蕤周小飞白阳姚佳维郭朝设
Owner BIOCYTOGEN JIANGSU CO LTD
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