Preparation method and application of humanized gene modification animal model

A humanized, non-human animal technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, plant genetic improvement, etc., can solve the problem of delayed response of memory T cells, reduce the risk of drug development, save money Time and cost, the effect of speeding up the development process

Active Publication Date: 2018-07-03
BIOCYTOGEN JIANGSU CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Delayed memory T cell responses in CD27-deficient mice compared with wild-type mice

Method used

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  • Preparation method and application of humanized gene modification animal model
  • Preparation method and application of humanized gene modification animal model
  • Preparation method and application of humanized gene modification animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1 Homologous Recombination Fragment Primer Design and PCR Amplification

[0133] According to the experimental design scheme, primers for amplifying seven homologous recombination fragments (A1, A2-1, A2-2, A3, B, C1, C2) were designed. The primer sequences are shown in Table 1.

[0134] Table 1 Homologous recombination fragments and their corresponding lengths and primer sequences

[0135]

[0136]

[0137] Using BAC as a template, 7 homologous recombination fragments were amplified using KOD-plus-polymerase. Among them, A1, A3, B, C1, and C2 select the BAC containing the mouse Cd27 genome as the template, and A2-1 and A2-2 select the BAC containing the human CD27 genome as the template. The specific PCR reaction system and reaction conditions are shown in Table 2.

[0138] Table 2PCR reaction system and reaction conditions

[0139]

[0140] The product fragments A1, A2-1, A2-2, A3, B, C1, and C2 obtained by PCR amplification are recovered and direc...

Embodiment 2

[0141] Example 2 Construction of Homologous Recombination Targeting Vector

[0142] Schematic diagram of comparison between mouse Cd27 gene and human CD27 gene figure 1 shown. The inventor designed figure 2 The targeting strategy is shown and the design of the targeting vector is shown. Mouse Cd27 (based on NM_001033126.2, namely SEQ ID NO: 15, and its corresponding protein expression NP_001028298.1, namely SEQ ID NO: 16) has a total of 6 exons. The child (all or part) is replaced with a human CD27 gene (based on NM_001242.4, namely SEQ ID NO: 17, and its corresponding protein expression NP_001233.1, namely SEQ ID NO: 18), and the neo gene is added after the 3'UTR with Screening of positive clones. The length of the 5' homology arm (SEQ ID NO: 19) is 4303bp, the length of the 3' homology arm is 4742bp (SEQ ID NO: 20), and the length of the human DNA fragment is 5810bp (SEQ ID NO: 21). The final desired CDS region, mRNA sequence and encoded protein sequence of the humaniz...

Embodiment 3

[0162] Example 3 Verification of carrier pDTA-down-ABC

[0163] Randomly select 4 pDTA-down-ABC clones, and use 3 sets of enzymes to perform digestion verification. Among them, 6817p+4629bp+3682bp+2952bp+2618bp+1786bp+1035bp should appear after digestion with BamHI, and should appear after digestion with XhoI 9976bp+5502bp+4037bp+2210bp+1477bp+317bp, after digestion with EcoRI, 8679bp+6589bp+5033bp+3218bp should appear. The enzyme digestion results were all in line with expectations, see Figure 5 , indicating that all plasmid digestion verification results are correct. Among them, the plasmids numbered 1-1 and 2-1 were verified to be correct by the sequencing company.

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Abstract

The invention relates to humanized gene modification of non-human animals, especially gene modification of rodents, and particularly gene modification of mice, and specifically relates to an establishment method of a humanized CD27 gene animal model and application thereof 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, to the construction method of a humanized CD27 genetically modified animal model and its application in biomedicine. Background technique [0002] Experimental animal disease models are indispensable research tools for the study of the etiology and pathogenesis of human diseases, and the development of prevention techniques and 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 replacement or replacement of homologous genes in ani...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N15/12C12N15/62C07K19/00A01K67/027
CPCA01K67/0278C12N15/85C07K14/70578A01K2267/03A01K2267/0331A01K2267/0387A01K2217/072A01K2227/105C07K2319/00C07K16/2878A61K2039/505G01N33/5011G01N33/5088G01N2333/70578C12N2015/8527A01K2207/15A01K2217/052
Inventor 沈月雷白阳黄蕤尚诚彰郭雅南张美玲
Owner BIOCYTOGEN JIANGSU CO LTD
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