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Preparation method and application of humanized gene modification animal model

An animal model and humanized technology, applied in botany equipment and methods, biochemical equipment and methods, plant genetic improvement, etc., to reduce the risk of drug development, speed up the research and development process, save time and cost

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, there are still many differences in the gene and protein sequences of humans and animals. Many human proteins cannot combine with homologous proteins of animals to produce biological activities, resulting in the results of many clinical trials inconsistent with the results of animal experiments.

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

[0177] Example 1 Construction of pT7-C-7 and pT7-C-18

[0178] 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.

[0179] Design and synthesize guide RNA sequences that recognize the 5' target site (sgRNA1-sgRNA12) and the 3' target site (sgRNA13-sgRNA24). Both the 5' target site and the 3' target site are located on the second exon of the mouse Ctla-4 gene, and the target site sequences of each sgRNA on Ctla-4 are as follows:

[0180] sgRNA-1 target site sequence (SEQ ID NO: 1): 5'-ggtcacctgtatggctgacatgg-3'

[0181] sgRNA-2 target site sequence (SEQ ID NO: 2): 5'-tgaaggttgggtcacctgtatgg-3'

[0182] sgRNA-3 target site sequence (SEQ ID NO: 3): 5'-acaggtgacccaaccttcagtgg-3'

[0183] sgRNA-4 target site sequence (SEQ ID NO: 4): 5'-gacccaaccttcagtggtgttgg-3' ...

Embodiment 2

[0215] Example 2 Construction of vector pClon-4G-CTLA-4

[0216]The partial coding sequence of exon 2 of the mouse Ctla 4 gene (Gene ID: 12477) (based on the transcript whose NCBI accession number is NM_009843.4 → NP_033973.2, its mRNA sequence is shown in SEQ ID NO: 34, The corresponding protein sequence is shown in SEQ ID NO: 35) is replaced with the corresponding coding sequence of human homologous CTLA4 gene (Gene ID: 1493) (based on the transcript of NCBI accession number NM_005214.4 → NP_005205.2, its mRNA sequence is shown in SEQ ID NO: 36, the corresponding protein sequence is shown in SEQ ID NO: 37), wherein, the comparison diagram of mouse CTLA 4 and human CTLA4 is shown in image 3 , the schematic diagram of the transformed humanized mouse CTLA4 gene finally obtained is shown in Figure 4 , the humanized mouse CTLA4 gene DNA sequence (chimeric CTLA4 gene DNA) is shown in SEQ ID NO:38:

[0217]

[0218] SEQ ID NO: 38 only lists the DNA sequence involved in the m...

Embodiment 3

[0238] Example 3 Verification of vector pClon-4G-CTLA-4

[0239] Randomly select 3 pClon-4G-CTLA-4 clones, and use 3 groups of enzymes for digestion verification, among them, HindIII should appear 1070bp+5250bp, BamHI+XhoI should appear 2348bp+3972bp, EcoRI+SmaI should appear 2818bp+3502bp, enzyme All results are as expected, see Figure 6 , in which the plasmids numbered 1 and 2 were verified to be correct by the sequencing company. 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 CTLA-4 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 CTLA-4 gene animal model and its application in biomedicine. Background technique [0002] Cancer is currently one of the diseases that cause the highest mortality in humans. According to the statistics of the World Health Organization, in 2012, the incidence and death of malignant tumors in the world reached 14 million and 8.2 million, and the number of newly diagnosed cancer cases in China was 3.07 million, and the death toll was 2.2 million. In recent years, the development of antibody drugs targeting immune checkpoints has been considered as a potential target to overcome various types of cancer. In traditional drug research and development, in vitro drug efficacy screening is adopted. This screening method cannot simulate the in vivo environment (such as tumor microenvironment, stromal...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N15/113C12N15/12C07K14/705A01K67/027
CPCA01K67/0276A01K67/0278A01K2217/072A01K2217/075A01K2227/105A01K2267/03C07K14/70521C12N15/113C12N15/8509C12N15/907C12N2310/10C12N2800/107A01K2267/0331A61K49/0008A61K2039/505A61K2039/507A61K2039/545A61K2039/55A61P35/00C07K16/2818C07K2317/21C07K2317/76C07K2319/00C12N15/1138C12N2320/11C12N2310/20
Inventor 沈月雷郭雅南白阳姚佳维尚诚彰黄蕤
Owner BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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