Cancer-causing mouse model as well as establishment method and application thereof

A mouse model and technology for establishing a method, applied in the field of animal models, can solve problems such as genetic instability, unfriendliness, and loss of foreign genes in animal models, and achieve the goal of avoiding host gene inactivation, overcoming technical difficulties, and improving expression levels Effect

Active Publication Date: 2022-03-04
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The insertion site is also directly related to the genetic stability of the model. Some host sites are "unfriendly" to the insertion of foreign genes, which often cause cutting and exclusion of foreign genes, resulting in the loss of foreign genes and genetic instability of animal models. happens sometimes
[0008] In summary, it is a difficult technology in this field to realize the insertion of a large fragment of foreign gene of more than 20kb into a specific site in the mouse genome, and to obtain an animal model with normal expression and stable inheritance, and there are few similar reports so far.

Method used

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  • Cancer-causing mouse model as well as establishment method and application thereof
  • Cancer-causing mouse model as well as establishment method and application thereof
  • Cancer-causing mouse model as well as establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] A carcinogenic mouse model constructed by:

[0064] 1. Gene synthesis

[0065] According to the NCBI database query, the complete sequence of the single-copy human proto-oncogene HRAS was obtained. Through gene synthesis, a single copy of the HRAS sequence was obtained, the sequence of which is shown in SEQ ID NO.1, and inserted into the cloning vector; through in vivo BAC (bacterial artificial chromosome) recombination and screening, a cloning vector containing two copies of the HRAS gene was obtained, Realize two copies in series.

[0066] After direction identification, clones with the correct head-to-tail tandem direction were obtained; through BAC rescue again, the third copy of the HRAS gene was linked to it, and after direction identification, the cloning vector with the correct connection direction and the connection of 3 fragments was selected to form a clone. The 21kb large fragment of the human proto-oncogene HRAS gene contains three copies, and retains the...

Embodiment 2

[0124] In this example, the mouse model obtained above is verified.

[0125] 1. Carcinogenicity verification experiment design

[0126] The test protocol was designed with reference to the technical guidelines on the necessity of drug carcinogenicity tests issued by NMPA, and the S1A and S1B guidelines issued by ICH (REF1-4), and the test items were implemented in accordance with the content of drug carcinogenicity evaluation under GLP conditions.

[0127] The test consisted of a vehicle control group and a positive test group respectively, the dosage was 75 mg / kg, the administration method was a single intraperitoneal injection, and the positive carcinogen was methylnitrosourea (MNU).

[0128] The positive test group was divided into two groups: (1) transgenic mouse MNU group (KI.C57-HRAS, or KI.C57-rasV2.0), a total of 50 mice, 25 male and female; (2) wild type Mouse MNU group (C57BL / 6), a total of 50 mice, 25 male and 25 male;

[0129] Vehicle control group: (1) Transgeni...

Embodiment 3

[0158] Carcinogenicity experiments were performed on the mouse model prepared in Example 1 to verify.

[0159] 1. Verification example 1

[0160] Select KI.C57-rasV2.0 model mice, half male and half male, and divide them into 2 groups by simple randomization according to sex and body weight. Group 1 was a 0.5% CMC-Na negative control group, with 25 animals per sex. Group 2 is the MNU positive control group (N-methylnitrosourea, the dose is 80 mg / kg), with 15 animals per sex. The first group was administered orally once a day for 26 weeks. On the first day, group 2 was intraperitoneally injected with methylnitrosourea (MNU), once.

[0161] The experimental results showed that: in the 0.5% CMC-Na negative control group, all male animals survived to the planned dissection day, and no unplanned death occurred; 1 female animal died on D79, and the rest survived to the planned dissection day. In the positive control MNU group, the first death time of male animals was D53, and th...

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Abstract

The invention relates to a carcinogenic mouse model and an establishment method and application thereof, and belongs to the technical field of animal models. The mouse model is obtained by transferring a vector containing a human protooncogene c-Ha-ras (HRAS) gene sequence fragment into C57BL/6 mouse embryonic stem cells, performing blastocyst injection, transplanting into a receptor mouse and performing screening. According to the mouse model, a stem cell targeting technology is adopted, a transgenic vector for highly expressing human proto-oncogene is established and subjected to fixed-point insertion, the mouse model is obtained through construction, C57BL/6 stem cells are directly used as a mouse background, a fixed-point insertion model with the pure C57BL/6 background can be directly obtained without long backcross, and the obtained mouse model is more uniform and better in result consistency.

Description

technical field [0001] The invention relates to the technical field of animal models, in particular to a carcinogenic mouse model and its establishment method and application. Background technique [0002] An animal model of human disease refers to an animal with a simulated phenotype of a human disease established in medical science research. The use of animal models of diseases can be traced back to the 19th century. The use of animal models can simplify complex problems, answer some life phenomena and laws, and help people start research on biological problems that seem impossible to start with. In the genome sequencing and post-genome era, since a large number of genes related to human genetic diseases have been discovered through sequencing, the study of the functions and pathogenic mechanisms of these genes is inseparable from animal models. In history, many well-known scientists and figures have promoted the development of animal models of diseases, including Aristot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/877C12N15/12A61D19/04A01K67/027A61K49/00
CPCC12N15/8509C12N15/8775C07K14/82A61D19/04A01K67/0278A61K49/0008A01K2207/15A01K2217/072A01K2227/105A01K2267/0331
Inventor 范昌发刘甦苏霍桂桃杨艳伟赵皓阳王三龙耿兴超孙晓炜谷文达翟世杰李琳丽吴勇曹愿
Owner NAT INST FOR FOOD & DRUG CONTROL
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