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Construction method for CD137-gene-modified humanization animal model and application thereof

A construction method and gene modification technology, applied in the field of animal genetic engineering and genetic modification, can solve problems such as hindering clinical development

Active Publication Date: 2019-05-10
GEMPHARMATECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the data of Urelumab in Phase 1 and Phase 2 showed severe clinical adverse reactions of grade 3-4, which hindered its clinical development

Method used

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  • Construction method for CD137-gene-modified humanization animal model and application thereof
  • Construction method for CD137-gene-modified humanization animal model and application thereof
  • Construction method for CD137-gene-modified humanization animal model and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of CD137 Humanized Targeting Homologous DNA Donor

[0062]In the present invention, after comparing human CD137 and mouse CD137 sequences, the complete extracellular region of human CD137 is selected to replace the complete extracellular sequence of mouse CD137, and the signal peptide, transmembrane region and intracellular region of mouse CD137 are retained. The sequence of the extracellular region of the source CD137 gene is shown in SEQ ID No:1, and the sequence of the extracellular region of the human CD137 gene is shown in SEQ ID No:2. Based on the human BAC (RP11-1080K5) sequence, the CD137 extracellular region sequence was amplified, and the homology arms at both ends of the mouse extracellular region ( SEQ ID No:27 and SEQ ID No:28), were connected with pMD18T to construct a homologous DNA donor, and the full sequence is shown in SEQ ID No:3.

Embodiment 2

[0063] Example 2 CD137 humanized mouse sgRNA screening

[0064] Design sgRNA targeting murine sequence in the humanized replacement region. Design and synthesize the recognition 5' end target site and 3' end target site, and construct sgRNA expression vector. The sgRNA recognition sites at both ends are respectively located at the two ends of the extracellular region of the mouse CD137 gene, and the target site sequences of each sgRNA on CD137 are shown in Table 1. Each sgRNA sequence was cloned into the pUC57kan-T7-delG vector to construct a puc57-sgRNA plasmid) (taking 000060-CD137-5S1 as an example, see SEQ ID No: 8 for the sequence of puc57-sgRNA-5S1).

[0065] Table 1 sgRNA sequence

[0066] sgRNA name

[0067] sgRNA transcription preparation method: use PrimerStar or PrimerStar Max system, sgRNA-F, sgRNA-R as primers, and correctly sequenced puc57-sgRNA plasmid as template to perform PCR, and purify PCR products to prepare sgRNA transcription preparation temp...

Embodiment 3

[0076] Example 3 Establishment of CD137 humanized mouse model

[0077] The sgRNA screened in Example 2 and the CD137 humanized targeting homologous DNA donor designed in Example 1 were injected into 0.5-day-old C57BL / 6 mouse fertilized eggs with the homologous DNA donor and Cas9 / sgRNA system. Transplanted into 0.5-day pseudopregnant female mice, after the mice were born, the target mice (F0) were screened out through genetic identification.

[0078] Genotyping of the F0 generation of humanized mice. Two pairs of primers were used to identify the sequence at both ends of the target in the homologous DNA donor and the sequence in the homologous DNA donor by using two pairs of primers for the obtained mouse tail genomic DNA of the F0 mouse. Primer 000060-huCD137-5'- tF1 / 000060-huCD137-tR1 are respectively located outside the 5' homologous arm and within the human fragment of the homologous DNA donor. If the PCR product is amplified by this pair of primers, it indicates that the ...

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Abstract

The invention provides a preparing method for a CD137-gene-modified humanization animal model based on a CRISPR / Cas9 technology. The CD137-gene-modified humanization animal model which has a completeimmune system and can effectively evaluate a CD137-resisting antibody is constructed. Further, through the CD137-gene-modified humanization animal model, a preclinical-screening CD137 humanization mouse pharmaceutical-effect evaluation platform capable of being used for the CD137-resisting antibody is constructed, a reasonable feeding method of the CD137-resisting antibody can be designed according to the pharmaceutical-effect evaluation platform, and a more-accurate preclinical pharmaceutical-effect evaluation method is provided for candidate CD137-resisting antibodies.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to a method for constructing a humanized animal model of CD137 gene modification based on RISPR / Cas9 technology and its application in biomedicine. Background technique [0002] Complex biological processes often require in vivo analysis, and many important research advances have used mice as models for studying various biological systems. In vivo studies of human biology are severely constrained ethically and technically, and animal models are increasingly required for in vivo studies of human cells, tissues, and organs. Currently, scientists have developed a variety of humanized mice or human-mouse chimeras to overcome these limitations and have now become important tools for in vivo studies of human cells and tissues. [0003] During the research and development of clinical drugs, mice are widely used in the preclinical safety and ef...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/90A01K67/027
Inventor 赵静琚存祥马秀英张明坤侯欢欢高翔
Owner GEMPHARMATECH CO LTD
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