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 pr

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

Example Embodiment

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

[0178] The target sequence determines the specificity of sgRNA targeting and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors.

[0179] Design and synthesize a guide RNA sequence that recognizes the 5'end target site (sgRNA1-sgRNA12) and the 3'end target site (sgRNA13-sgRNA24). The 5'end target site and the 3'end target site are both located on the second exon of the mouse Ctla-4 gene. The target site sequence of each sgRNA on Ctla-4 is 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’

[0184] sgRNA-...

Example Embodiment

[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 with NCBI accession number NM_009843.4→NP_033973.2, the mRNA sequence is shown in SEQ ID NO: 34, The corresponding protein sequence is shown in SEQ ID NO: 35) and the corresponding coding sequence of the human homologous CTLA4 gene (Gene ID: 1493) is replaced (based on the transcript with NCBI accession number NM_005214.4→NP_005205.2, and its mRNA sequence is shown in SEQ ID NO: 36, the corresponding protein sequence is shown in SEQ ID NO: 37), among which, the comparison diagram of mouse Ctla 4 and human CTLA4 is shown in image 3 , The final modified humanized mouse CTLA4 gene diagram 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 modification, and the underlined ar...

Example Embodiment

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

[0239] Randomly select 3 pClon-4G-CTLA-4 clones, and use 3 sets of enzymes for restriction enzyme digestion. Among them, HindIII should appear 1070bp+5250bp, BamHI+XhoI should appear 2348bp+3972bp, EcoRI+SmaI should appear 2818bp+3502bp, enzyme All results are in line with expectations, see Image 6 , The plasmids numbered 1 and 2 were verified by the sequencing company to be correct. Select plasmid 1 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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