Method for constructing in-situ liver cancer animal model

An animal model, in situ technology, applied in the field of animal models, can solve the problems of being unsuitable for the immune system, high technical requirements, and high mortality in modeling, and achieve the effect of shortening the tumor formation time and improving the tumor formation rate.

Active Publication Date: 2022-05-27
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of liver cancer research, the current experimental liver cancer animal models still have many deficiencies: (1) The chemical drug-induced model has a model-making time of more than one year, and has defects such as a high mortality rate; (2) The subcutaneous cell transplanted tumor model The environment of clinical tumor growth cannot be simulated; (3) The implantation process of the orthotopic tumor model is cumbersome and requires high technical requirements. In addition, the implantation model mostly uses immunodeficient mice, which is not suitable for the study of the immune system
However, the animal models produced by these gene editing alone are of a single variety, and the tumor formation time generally takes more than 6 months, which is difficult to meet the needs of different researchers to explore animal tumors caused by different gene editing backgrounds.

Method used

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  • Method for constructing in-situ liver cancer animal model
  • Method for constructing in-situ liver cancer animal model
  • Method for constructing in-situ liver cancer animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Target gene sgRNA design.

[0039]According to the gene sequence of PTEN or AXIN1 or TRP53, the sgRNA of PTEN or AXIN1 or TP53 was designed and synthesized according to the sgRNA design website (http: / / crispr.mit.edu / ). The specific sgRNA sequences of each gene are as follows. Three sgRNAs were designed for each gene and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0040] Table 1 sgRNA

[0041]

[0042]

[0043] PCR primers were designed according to the sgRNA sequence, and the cellular genomic DNA was used as the template for PCR amplification, and the PCR products were sequenced to determine whether there was a SNP site in the target genome position bound by the sgRNA, and to determine the possibility of off-target. details as follows:

[0044] 1.1Trp53 gene

[0045] (1) Trp53 target design

[0046] Table 1

[0047]

[0048] (2) SNP detection primers in Trp53 target region

[0049] Table 2

[0050]

[0051] (3) PCR reaction system ...

Embodiment 2

[0137] Construction of pX330-U6-sgRNA(PTEN or Axin1 or TP53)-Chimeric_BB-CBh-hSpCas9-T2A-mCherry plasmid

[0138] 1. Transformation of pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid

[0139] The T2A-mCherry gene was cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (see the plasmid map Figure 11 )middle.

[0140] (1) The pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid was digested with EcoRI enzyme to be linear, and electrophoresis was used to observe whether the digestion result was linear.

[0141] (2) PCR amplification of T2A-mCherry target gene

[0142] The primers are as follows, and the end of the primer introduces an EcoRI restriction site,:

[0143] Table 13

[0144]

[0145] Primer description: It contains exchange paired bases, restriction enzyme sites, and contains part of the 5'-end sequence of the target gene for PCR to fish for the target gene.

[0146] Using the original plasmid with T2A-mCherry as a template, the above-mentioned primers were used to amplify ...

Embodiment 3

[0183] According to the above examples 1 and 2, 3 sgRNAs of PTEN or AXIN1 or TP53 gene have been successfully cloned into the CRISPR / Cas plasmid system, and the mouse liver cancer cells were transfected with T7 endonuclease I (T7E1) enzyme. Excision activity screening produces sgRNAs with stronger editing ability for target genes. The specific operation steps are as follows:

[0184] (1) Identification of sgRNA editing ability by T7E1 digestion

[0185] Enzyme digestion system: purified product 4-7μL (purified product at 500ng), Detecase Buffer 2μL, Detecase 0.5μL, ddH 2 O make up volume 20 μL. After 60 min of reaction at 37°C, 2 μL of Top Buffer was added to the above 20 μL system immediately.

[0186] (2) Trp53 gene

[0187] Table 21 sgRNA editing ability T7E1 digestion

[0188]

[0189] PCR reaction system for SNP detection in Trp53 target region: 1 μL of upstream and downstream primers (10 μM); 25 μL of 2×Taq Plus Master Mix; 2 μL of Genomic DNA (>200ng / μL); ddH 2...

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Abstract

The invention discloses a method for constructing an in-situ liver cancer animal model, and belongs to the field of animal models. The sgRNA targeting AXIN1 gene, PTEN gene and TP53 gene and having good editing ability is obtained through screening, the nucleotide sequence of the sgRNA specifically targeting the AXIN1 gene is as shown in SEQ ID NO: 1-3, the nucleotide sequence of the sgRNA specifically targeting the PTEN gene is as shown in SEQ ID NO: 4-6, and the nucleotide sequence of the sgRNA specifically targeting the TP53 gene is as shown in SEQ ID NO: 7-9; the sgRNA is injected into the animal liver through a tandem gene editing technology to construct the in-situ liver cancer animal model. Experimental verification shows that a knockout model constructed by combining 2-3 cancer suppressor genes has obviously formed tumors in 3-4 months, and the highest tumor formation rate can reach 100%.

Description

technical field [0001] The present invention relates to the field of animal models, in particular to a method for constructing an orthotopic liver cancer animal model. Background technique [0002] The biopharmaceutical industry has always been a strategic emerging industry for national key development. The development of the biopharmaceutical industry is inseparable from innovative and cutting-edge basic scientific research in medicine and pharmacy. It is an important experimental tool for biological characteristics, pathogenesis, drug screening and disease treatment. It can be said that without experimental animals, there would be no development and progress of biomedicine today. In terms of liver cancer disease research, there are still many shortcomings in the current experimental liver cancer animal models: (1) the chemical drug-induced model has a modeling time of more than 1 year, and the high mortality rate of modeling; (2) subcutaneous cell transplantation tumor mod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N9/22C12N15/12C12N15/85C12N5/10A01K67/027
CPCC12N15/113C12N9/22C12N15/85C07K14/47A01K67/0276C12N2310/20A01K2217/075A01K2267/0331Y02A50/30
Inventor 周素芳
Owner GUANGXI MEDICAL UNIVERSITY
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