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Induced inflammatory cancer transformation mouse model as well as establishment method and application thereof

A mouse model and mouse technology, applied in chemical instruments and methods, biochemical equipment and methods, applications, etc., can solve the problems of living mouse models for a long time, shorten the time of tumor formation, promote liver tumor formation, The effect of improving modeling efficiency

Active Publication Date: 2021-06-25
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although this document 1 combined the use of hydrodynamic gene transfection technology and Sleeping Beauty transposase to integrate the transfected target gene into the mouse chromosome to achieve sustained expression of the target gene in the mouse liver, the use of the document 1 Methods It still takes a long time to construct a live mouse model, usually about 3 months

Method used

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  • Induced inflammatory cancer transformation mouse model as well as establishment method and application thereof
  • Induced inflammatory cancer transformation mouse model as well as establishment method and application thereof
  • Induced inflammatory cancer transformation mouse model as well as establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of lentiviral recombinant plasmids knocking out tumor suppressor genes pten and p53 in mice

[0040] 1.1. According to the enzyme digestion system shown in Table 1 below, use the restriction endonuclease BsmBI to the lentiCRISPR v2 vector ( figure 1 The map of the vector is shown) to carry out enzyme digestion and make it linearized as the backbone vector of the lentiviral recombinant plasmid for knocking out the tumor suppressor gene pten and p53 of mice; wherein the enzyme digestion conditions are: at 55°C After 15 minutes of enzyme digestion reaction, the enzyme was inactivated at 80°C for 20 minutes, and the digested product was subjected to 1% agarose electrophoresis. The results were as follows: figure 2 As shown, it can be seen that there is a band at 2kb, that is, the target linearized lentiCRISPR v2 vector is obtained, and the target vector band is recovered and stored at -20°C for later use.

[0041] Table 1: Restriction enzyme BsmBI ...

Embodiment 2

[0053] Example 2: Construction of Induced Inflammatory Cancer Transformation Mouse Model

[0054] This example uses the lentiCRISPR-sgPten lentiCRISPR-sgPten knockout mouse tumor suppressor gene pten knockout lentiCRISPR-sgPten knockout mouse tumor suppressor gene p53 lentiCRISPR-sgP53 knockout mouse tumor suppressor gene p53 obtained in the above-mentioned Example 1, and Sleeping Beauty The transposase expression plasmid pCMV / SB10, the recombinant plasmid pT3-EF1a-c-met overexpressing the c-met gene, and the recombinant plasmid pT3-N90-β-catenin overexpressing the Δ90-β-catein gene (the latter three were purchased from Since addgene, such as Figure 3 to Figure 5 , respectively showing the maps of the three plasmids, after the enrichment, a large number of plasmids were extracted, and stored at -20°C for future use) to establish a mouse liver cancer model, which specifically included the following operations: Using the hydrodynamic gene transfection method, the C57BL / 6 mouse ...

Embodiment 3

[0058] Example 3, Formation Verification of Induced Inflammatory Cancer Transformation Mouse Model

[0059] After the 7th week of transfection (that is, 6 weeks later), the mice were sacrificed to observe the tumor formation in the liver of the mice. The mice in the experimental group were transfected with pCMV / SB10 (5 μg), pT3-EF1a-c-met (10 μg), pT3 -Mixed plasmids of N90-β-catenin (10 μg), lentiCRISPR-sgPten (10 μg) and lentiCRISPR-sgP53 (10 μg), mice in the control group were transfected with lentiCRISPRv2 (20 μg) and pCMV / SB10 (5 μg) plasmids, HE staining Observe the pathological changes of mouse liver:

[0060] (1) Take a mouse liver tissue sample with a thickness of about 3mm, dehydrate with gradient alcohol at 70%, 80%, 95%, and 100% for 30 minutes each, two bottles of xylene for 20 minutes each, and two cylinders of paraffin wax for 12 minutes each. After embedding in paraffin, the sections were about 4 μm thick.

[0061] (2) Hematoxylin eosin (HE) staining: ① Dewax...

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Abstract

The invention discloses an induced inflammatory cancer transformation mouse model as well as an establishment method and application thereof, and belongs to the technical field of biology. The method provided by the invention comprises the following steps: jointly dissolving recombinant plasmids of overexpressed exogenous oncogene, sleeping beauty transposase expression plasmids and CRISPR / Cas9 recombinant plasmids capable of knocking out target cancer suppressor genes into normal saline, and injecting the normal saline into caudal veins of mice at a speed of less than 5 seconds. The induced inflammatory cancer transformation mouse model in which the exogenous oncogene is continuously overexpressed in the mouse liver, the expression of the cancer suppressor gene in the mouse liver is continuously knocked down and the mouse liver is tumorigenic can be rapidly established through one-step operation, and the induced inflammatory cancer transformation mouse model can be used for screening primary cancer early immune markers and / or anti-cancer drugs.

Description

technical field [0001] The invention belongs to the field of biological technology, and specifically relates to an induced inflammatory cancer transformation mouse model and its establishment method and application, especially relates to an induced type in which the expression of two oncogenes is overexpressed and the expression of two tumor suppressor genes is knocked down. Inflammatory cancer transformation mouse model and its rapid establishment method, and the application of the model in screening cancer (such as primary liver cancer) early immunodiagnostic markers and / or anticancer drugs. Background technique [0002] Hepatocellular carcinoma (HCC) is the most common primary malignant lesion of the liver, and its mortality rate is gradually increasing. The onset of HCC is hidden, and many patients are already in the middle and advanced stages when they are diagnosed, and they lose the opportunity for surgical treatment. There are few targeted therapy drugs and the progn...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12C12N15/54A01K67/027
CPCC12N15/8509C07K14/82C12N9/1241A01K67/0276A01K2217/075A01K2227/105A01K2267/0331C12N2310/20
Inventor 詹林盛吕丽萍王小慧张玉龙曹相宜周欠欠马平孙苏静
Owner ACADEMY OF MILITARY MEDICAL SCI
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