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Preparation method and application of humanized gene modification animal model

An animal model, humanized technology, applied in the establishment of humanized genetic animal models, can solve problems such as inapplicability, save time and cost, speed up the research and development process, and reduce the risk of drug development.

Active Publication Date: 2018-03-20
BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the significant difference between human physiology and animal physiology, the experimental results obtained by using animal models sometimes cannot be applied to humans, while humanized animal models can well "replicate" certain human disease characteristics. It is used as an in vivo surrogate model for human disease research, and for in vivo drug efficacy testing experiments in preclinical drug development

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

[0186] Construction of embodiment 1 pT7-B2-6, pT7-B2-10

[0187] The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors.

[0188] Design and synthesize guide RNA sequences that recognize 5' target sites (sgRNA1-sgRNA8) and 3' target sites (sgRNA9-sgRNA17). The target site sequence of each sgRNA on Tigit is as follows:

[0189] sgRNA-1 target site sequence (SEQ ID NO:1): 5'-ctgaagtgacccaagtcgactgg-3'

[0190] sgRNA-2 target site sequence (SEQ ID NO:2): 5'-ctgctgcttccagtcgacttggg-3'

[0191] sgRNA-3 target site sequence (SEQ ID NO:3): 5'-ggccattattagtgttgacctgg-3'

[0192] sgRNA-4 target site sequence (SEQ ID NO:4): 5'-ccccaggtcaacactataaatgg-3'

[0193] sgRNA-5 target site sequence (SEQ ID NO:5): 5'-caggcacgatagatacaaagagg-3'

[0194] sgRNA-6 target site sequence...

Embodiment 2

[0220] The construction of embodiment 2 vector pClon-4G-TIGIT

[0221]The partial coding sequence of exon 2 of the mouse Tigit gene (Gene ID: 100043314) (based on the transcript of NCBI accession number NM_001146325.1 → NP_001139797.1, its mRNA sequence is shown in SEQ ID NO: 27, corresponding to The protein sequence shown in SEQ ID NO: 28) is replaced with the corresponding coding sequence of human source TIGIT gene (Gene ID: 201633) (based on the transcript whose NCBI accession number is NM_173799.3 → NP_776160.2, its mRNA sequence is shown in SEQ ID NO : 29, the corresponding protein sequence is shown in SEQ ID NO: 30), and the comparative schematic diagram of mouse Tigit and human TIGIT gene structure is shown in image 3 , the schematic diagram of the transformed humanized mouse TIGIT gene finally obtained is shown in Figure 4 . The humanized mouse TIGIT gene DNA sequence (chimeric TIGIT gene DNA) is shown in SEQ ID NO:31:

[0222]

[0223] SEQ ID NO: 31 only lists...

Embodiment 3

[0251] Example 3 Verification of vector pClon-4G-TIGIT

[0252] Randomly select 3 pClon-4G-TIGIT clones, and use 3 groups of enzymes for enzyme digestion verification. Among them, XbaI+XhoI should appear 4766bp+2011bp, HindIII+ScaI should appear 4233bp+1283bp+729bp+524bp+8bp, BamHI+BglII should appear 4917bp+1860bp appeared, and the digestion results were all in line with expectations, see Figure 6 , where the plasmids numbered 1 and 2 were verified to be correct by sequencing company, and plasmid 1 was selected for subsequent experiments.

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Abstract

The invention relates to a humanized gene genetically modified non-human animal, particularly a genetically modified rodent, especially a genetically modified mouse, and in particular relates to a construction method of a humanized TIGIT gene animal model and application of the model in the biomedicine field.

Description

technical field [0001] This application relates to the establishment method and application of a humanized gene animal model, in particular, to a construction method based on a humanized TIGIT gene animal model and its application in biomedicine. Background technique [0002] Often, it is desirable to conduct experiments using human cells in the study of human disease. In particular, it is necessary to use human-derived gene expression models in studies to confirm the involvement of species-specific multiple drug-metabolizing enzymes, host-limited immune responses, and other diseases. [0003] 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. Common experimental animals include mice, rats, guinea pigs, hamsters (hamsters), rabbits, dogs, monkeys, pigs, fish, etc. However, there are still many differences in the gene and protein sequenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/113C12N15/12C07K14/705A01K67/027
CPCA01K67/0276A01K67/0278C07K14/70503C12N15/113C12N15/8509C12N15/907A01K2267/03A01K2217/072A01K2217/075A01K2227/105C12N2310/10C12N2800/107C12N15/90A01K2207/15A01K2267/0331C07K2319/00C12N2517/02A61K49/0008A01K2207/12C07K2319/03C12N2015/8572
Inventor 沈月雷白阳黄蕤郭雅南周小飞张美玲
Owner BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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