Induced inflammatory carcinoma transformation mouse model and its establishment method and application

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: 2022-07-12
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 carcinoma transformation mouse model and its establishment method and application
  • Induced inflammatory carcinoma transformation mouse model and its establishment method and application
  • Induced inflammatory carcinoma transformation mouse model and its establishment method and application

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. Use restriction endonuclease BsmBI to lentiCRISPR v2 vector ( figure 1 The map of the vector is shown), and it is linearized and used as a backbone vector for constructing a lentiviral recombinant plasmid knocking out tumor suppressor genes pten and p53 in mice; the restriction enzyme digestion conditions are: under the condition of 55 ℃ After the digestion reaction for 15 minutes, the enzyme was inactivated at 80°C for 20 minutes, and the digestion product was subjected to 1% agarose electrophoresis, and 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 future use.

[0041] Table 1: The restriction endonuclease BsmBI digests the lentiCRISPRv2 vector

[0042]

[0043] ...

Embodiment 2

[0053] Example 2: Construction of a mouse model of induced inflammatory carcinoma transformation

[0054] In this example, the lentiCRISPR-sgPten lentivirus recombinant plasmid lentiCRISPR-sgPten knocking out the tumor suppressor gene p5 in mice, the lentiCRISPR-sgP53 lentivirus recombinant plasmid lentiCRISPR-sgP53 knocking out the tumor suppressor gene p53 in mice, 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 (all the latter three were purchased) from addgene companies such as Figure 3 to Figure 5 , respectively show the maps of the three plasmids. After enrichment, the plasmids were extracted in large quantities, and stored at -20°C for later use) to establish a mouse liver cancer model, which specifically included the following operations: using the hydrodynamic gene transfection method, through C57BL...

Embodiment 3

[0058] Example 3. Verification of the formation of a mouse model of induced inflammatory carcinoma transformation

[0059] After the 7th week of transfection (ie 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 (5μg), pT3-EF1a-c-met (10μg), pT3 - Mixed plasmids of N90-β-catenin (10μg), lentiCRISPR-sgPten (10μg) and lentiCRISPR-sgP53 (10μg), control mice were transfected with lentiCRISPRv2 (20μg) and pCMV / SB10 (5μg) plasmids, HE staining method Observe the pathological changes of mouse liver:

[0060] (1) Take mouse liver tissue samples with a thickness of about 3 mm, dehydrate them with gradient alcohol for 70%, 80%, 95%, and 100% for 30 minutes each, two bottles of xylene for 20 minutes each, and two tanks of paraffin immersion for 12 minutes each. Sectioned after paraffin embedding, the thickness is about 4 μm.

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

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Abstract

The invention discloses a mouse model of induced inflammatory carcinoma transformation and its establishment method and application, and belongs to the field of biotechnology. The method provided by the present invention includes dissolving a recombinant plasmid overexpressing an exogenous oncogene, a Sleeping Beauty transposase expression plasmid, and a CRISPR / Cas9 recombinant plasmid capable of knocking out a target tumor suppressor gene together in physiological saline, and the amount is less than 5 It is injected into the tail vein of mice at a speed of seconds, and the exogenous oncogene can be rapidly established in one step to continuously over-express in the mouse liver, the expression of tumor suppressor genes in the mouse liver is continuously knocked down, and the mouse liver can be continuously overexpressed. The tumor-inducing inflammatory-cancer transformation mouse model can be used to screen early immune markers and / or anti-cancer drugs of primary cancer.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an inducible inflammatory-cancer transformation mouse model and a method for its establishment and application, in particular to an inducible type in which two oncogenes are overexpressed and two tumor suppressor genes are knocked down. Inflammation-cancer transformation mouse model and rapid establishment method thereof, and application of the model in screening early immunodiagnostic markers and / or anti-cancer drugs of cancer (such as primary liver cancer). Background technique [0002] Hepatocellular carcinoma (HCC) is the most common primary malignant liver disease, and its mortality rate is increasing gradually. The onset of HCC is insidious, and many patients are already in the middle and late stages at the time of diagnosis, losing the opportunity for surgical treatment. There are few targeted therapy drugs and the prognosis is relatively poor. Therefore, in order to improve ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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