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Preparation method and application of humanized gene modified 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 problems of limited use in researching gene signaling pathways, functions, and inability to use antibody drugs. , to speed up the research and development process, reduce the risk of drug development, save time and cost

Active Publication Date: 2018-04-24
BIOCYTOGEN JIANGSU CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above studies show that the existing BTLA-related model animals are mainly BTLA- / -deficient mice, whose use is limited to the study of gene signaling pathways, functions, etc., and cannot be used for antibody drug screening

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0254] Construction of embodiment 1 pT7-BTLA-1, pT7-BTLA-14

[0255] 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.

[0256] Design and synthesize sgRNA sequences that recognize the 5' target site (sgRNA1-sgRNA8) and the 3' target site (sgRNA9-sgRNA14). Both the 5' target site and the 3' target site are located on the second exon of the BTLA gene, and the target site sequences of each sgRNA on BTLA are as follows:

[0257] sgRNA-1 target site sequence: 5'-CAGTGCAACTTACTATTACG-3' (SEQ ID NO: 1)

[0258] sgRNA-2 target site sequence: 5'-CTCGTAATAGTAAGTTGCAC-3' (SEQ ID NO: 2)

[0259] sgRNA-3 target site sequence: 5'-GTGACTTGGTGTAAGCACAA-3' (SEQ ID NO: 3)

[0260] sgRNA-4 target site sequence: 5'-TCCAAACAGTCTGCCAGGAC-3' (SEQ ID NO: 4)

[0261] sgRNA-5 targe...

Embodiment 2

[0282] The construction of embodiment 2 vector pClon-4G-BTLA

[0283] The partial coding sequence of exon 2 of the mouse Btla gene (Gene ID: 208154) (based on the transcript whose NCBI accession number is NM_001037719.2 → NP_001032808.2, its mRNA sequence is shown in SEQ ID NO: 24, corresponding to The protein sequence shown in SEQ ID NO: 25) is replaced with the corresponding coding sequence of human BTLA gene (Gene ID: 151888) (based on the transcript of NM_181780.3 → NP_861445.3 whose NCBI accession number is, its mRNA sequence is shown in SEQ ID NO : 26, and the corresponding protein sequence is shown in SEQ ID NO: 27), and the comparative schematic diagram of mouse Btla and human BTLA gene structure is shown in image 3 , the schematic diagram of the transformed humanized mouse BTLA gene finally obtained is shown in Figure 4 . The humanized mouse BTLA gene DNA sequence (chimeric BTLA gene DNA) is shown in SEQ ID NO: 28:

[0284] GATGAAGAG TGTGATGTACAGCTTTATATAAAGAGA...

Embodiment 3

[0304] Example 3 Verification of vector pClon-4G-BTLA

[0305] Randomly select 3 pClon-4G-BTLA clones, and use 3 sets of enzymes for digestion verification. Among them, EcoRI should appear 5779bp+1043bp, SacI should appear 4130bp+2692bp, and ScaI should appear 5579bp+1243bp. The enzyme digestion results were all in line with expectations, see Image 6 , the results showed that all plasmid digestions were verified correctly. Plasmids numbered 1, 2, and 3 were verified to be correct by the sequencing company, and plasmid 2 was selected for subsequent experiments.

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Abstract

The invention relates to a gene modified non-human animal of a humanized gene, especially a gene modified rodent animal and in especially a gene modified mouse, and in particular relates to a construction method of the humanized BTLA gene animal model, a subsequently generated animal and an application of the animal model in the field of bio-medicine.

Description

technical field [0001] This application relates to the establishment method and application of a humanized genetically modified animal model, specifically, the construction method based on a humanized BTLA genetically modified animal model, the resulting animal and its application in biomedicine. Background technique [0002] Immunotherapy, which activates the immune system to attack and kill cancer cells, is an important area of ​​cancer research and has been used in clinical treatment for nearly a decade. Studies have shown that targeting the inhibitory receptors of T cells has a significant therapeutic effect and is currently the most successful field of targeted gene therapy. Among them, monoclonal antibodies targeting CTLA-4 and PD-1 / PD-L1 have achieved definite curative effects, and more new drugs targeting other inhibitory receptors are entering or have entered clinical research. [0003] B. T lymphocyte attenuator (B and T lymphocyte attenuator, BTLA) is expressed o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/113C07K14/705C12N15/12A01K67/027
CPCA01K67/0276A01K67/0278A01K2207/15A01K2217/075A01K2227/105A01K2267/03C07K14/70521C12N9/22C12N15/113C12N15/8509C12N15/907C12N2310/10C12N2800/107
Inventor 沈月雷白阳郭雅南黄蕤周小飞张美玲
Owner BIOCYTOGEN JIANGSU CO LTD
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