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Construction method and application of humanized CCR2 gene modified animal model

A technology of genetic modification, construction method, applied in the field of biology

Active Publication Date: 2020-09-22
SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The screening and preclinical tests of human CCR2 target drugs need to be evaluated in animal models, but due to the difference in species, the homology between human CCR2 and mouse Ccr2 protein is only 68%, and the ones screened in mice Targeted drugs are not fully applicable to humans, and drugs targeting human CCR2 protein may not necessarily recognize mouse Ccr2 protein

Method used

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  • Construction method and application of humanized CCR2 gene modified animal model
  • Construction method and application of humanized CCR2 gene modified animal model
  • Construction method and application of humanized CCR2 gene modified animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 sequence design

[0065] Both the mouse Ccr2 gene and the human CCR2 gene contain multiple transcripts, and the sequence design in this example is mainly described using one of the transcripts as an example. That is, the main part of exon No. 3 of the mouse Ccr2 gene (NCBI Gene ID: 12772) (based on the transcript whose NCBI accession number is NM_009915.2 → NP_034045.1, its mRNA sequence is shown in SEQ ID NO: 1, The corresponding protein sequence is shown in SEQ ID NO: 2) is replaced with the corresponding fragment of human CCR2 gene (Gene ID: 729230) (based on the transcript whose NCBI accession number is NM_001123396.3 → NP_001116868.1, its mRNA sequence is as SEQ ID NO: 3, and the corresponding protein sequence is shown in SEQ ID NO: 4), wherein, the comparison diagram of mouse Ccr2 and human CCR2 genes is shown in figure 1 , the finally obtained transformed humanized mouse CCR2 gene schematic diagram is shown in figure 2 , the humanized mouse CCR2 g...

Embodiment 2

[0071] Example 2 Design and Construction of Recombinant Vector PBR322-CCR2

[0072] According to the sequence design, the inventor has further designed such as image 3 Targeting scheme shown and vector containing 5' homology arm, human CCR2 gene fragment, 3' homology arm. Wherein the 5' homology arm (SEQ ID NO: 9) is nucleotide 124102766-124105684 of NCBI accession number NC_000075.6, and the 3' homology arm (SEQ ID NO: 10) is NCBI accession number NC_000075. The nucleotides 124106806-124110723 of 6, and the human CCR2 (SEQ ID NO: 11) gene fragment are nucleotides 46357528-46358610 of NCBI accession number NC_000003.12.

[0073] The construction process of the vector is as follows: design the upstream primers for amplifying the three homologous recombination fragments (LA, KI, RA), the matching downstream primers and related sequences. Among them, the 5' homology arm corresponds to the LA fragment, the human CCR2 gene fragment corresponds to the KI fragment, and the 3' homo...

Embodiment 3

[0084] Example 3 Verification of carrier PBR322-CCR2

[0085] Randomly select 5 PBR322-CCR2 clones, and use the restriction endonuclease EcoRI to carry out enzyme digestion verification. The digested product should have a fragment size of 5614bp+4433bp+2759bp+1038bp in electrophoresis. For enzyme digestion results, see Figure 4 , the results of plasmid digestion were in line with expectations, indicating that the verification results of plasmid digestion were correct. The plasmid was verified to be correct by the sequencing company, and subsequent experiments were carried out.

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Abstract

The invention provides a construction method and application of a humanized CCR2 gene modified animal model, and relates to the technical field of biology. According to the humanized CCR2 gene modified animal model constructed by the invention, the research progress of the fields related to the human CCR2 gene or protein can be accelerated; for example, the humanized CCR2 gene modified animal model is used for replacing a human to test a drug; and an effective model and a powerful tool are provided for a preclinical experiment of a CCR2 target drug. Preferably, a CRISPR / Cas9 gene editing technology is utilized in the invention; a mouse-derived Ccr2 gene is replaced with a human-derived CCR2 gene, so that a mouse model capable of interacting with an anti-human CCR2 antibody is constructed;compared with a common mouse, humanized transformation of key target molecules is achieved; and the mouse model can be used for screening and evaluating drugs for human CCR2 genes, and is a very idealpreclinical drug test model.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a humanized CCR2 genetically modified animal model and its application. 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. Common experimental animals include mice, rats, guinea pigs, hamsters (hamsters), rabbits, dogs, monkeys, pigs, fish, etc. Although the vast majority of human genes have homologous genes in animals, there are still many differences between humans and animals in gene sequences and protein sequences, resulting in many drugs targeting human targets that cannot be combined with homologous proteins in animals. Drug efficacy, which is especially significant in macromolecular antibody drugs, because macromolecular antibody drugs have high specificity, and a single amino acid di...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/85C12N15/877C12N5/10C12N5/071C12N15/12A01K67/027A61K49/00
CPCC12N15/907C12N15/8509C12N15/8775C07K14/723A01K67/0278C12N5/0604C12N5/0602A61K49/0008C12N2800/107A01K2217/072A01K2227/105A01K2267/0331C12N2510/00C12N2503/02C12N2517/02
Inventor 费俭孙瑞林王津津慈磊
Owner SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV
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