Method for manufacturing adjustable liver injury animal model and special carriers thereof

An expression vector and eukaryotic expression vector technology, applied in the production of regulated animal models of liver injury and the field of dedicated vectors, can solve the problems of TRE downstream gene transcriptional activation blocking, inability to bind, etc., to reduce difficulty and improve survival rate effect

Active Publication Date: 2009-10-07
PEKING UNIV +1
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Problems solved by technology

In this way, contrary to the working principle of Tet O-Tet R mentioned above, when tetracycline is lacking, tTA protein binds to Tet O and activates the transcription of genes downstream of TRE; and when tetracycline is added, the high affinity of tetracycline and Tet R makes tTA protein A conformational change occurs, unable to bind Tet O, thereby blocking the transcriptional activation of genes downstream of TRE

Method used

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  • Method for manufacturing adjustable liver injury animal model and special carriers thereof
  • Method for manufacturing adjustable liver injury animal model and special carriers thereof
  • Method for manufacturing adjustable liver injury animal model and special carriers thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0084] Example 1, Construction of Tet-on Regulated Recombinant Vector for the Specific Expression of uPA Gene in Mouse Liver

[0085] There are two recombinant vectors that Tet-on regulates the specific expression of uPA gene in mouse liver, one is the recombinant expression vector pTRE-uPA carrying the response element TRE and uPA gene expressed by Tet-on, and the other is carrying the mouse The recombinant vector pAlb-rtTA of albumin (Albumin) promoter and Tet-on expression regulatory element rtTA gene. In pAlb-rtTA, the rtTA gene is located under the Albumin promoter and is regulated by it, and is specifically expressed in the mouse liver; and the change of the concentration of rtTA in the liver can regulate the expression of the prourokinase activator (uPA) gene.

[0086] The specific construction methods of pTRE-uPA and pAlb-rtTA are as follows:

[0087] 1. Construction of pTRE-uPA plasmid vector and preparation of DNA samples for prokaryotic injection

[0088] The cDNA...

Embodiment 2

[0240] Embodiment 2, making and breeding of transgenic mice

[0241] 1. Production and identification of transgenic mice

[0242] 1. Production of transgenic mice

[0243] ① Select 4-5 weeks old female ICR mice with closed vaginal opening as donors, and inject 10 IU of pregnant horse serum (PMSG) intraperitoneally into each mouse at around 3:00 pm.

[0244] ②47-48 hours later, each mouse was intraperitoneally injected with 10 IU of human chorionic gonadotropin (HCG), and caged with normal male mice to provide fertilized eggs. Another number of female mice of appropriate age (more than 2 months old) were selected, and the female mice in estrus with flushed vaginal orifice were selected as recipients, and they were caged with ligated male mice.

[0245] ③Observe the donor and recipient before 9:00 a.m. on the next day. Those who have sperm plugs take out spares, and the recipient cages are taken out for isolation measures.

[0246] ④ Around 10:30, the donor was executed by ne...

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Abstract

The invention discloses a method for manufacturing an adjustable liver injury animal model and special carriers thereof. The invention provides two special carriers; one is obtained by inserting DNA fragments formed by sequentially connecting tetracycline reacting factors, promoters and pro-urokinase activator coding genes of a Tet-off / Tet-on gene expression system from the upper stream to the lower stream into the multiple cloning sites of an eukaryotic expression carrier; and the other one is obtained by inserting DNA fragments formed by sequentially connecting liver special promoters and antisense tetracycline activator coding genes of a Tet-on gene expression system from the upper stream to the lower stream into the multiple cloning sites of an eukaryotic expression carrier. The two special carriers re implanted into the animal bodies for obtaining a double-positive transgenic animal model, and the expression time and the expression quantity of uPA genes in the liver cells of the animal model are adjusted through the induction of doxycline, thereby the time and the degree of the liver injury can be manually controlled according to demands.

Description

technical field [0001] The invention relates to a production method of an adjustable liver injury animal model and a special carrier thereof. Background technique [0002] Hepatitis C virus (HCV) is one of the main pathogens causing chronic hepatitis and seriously endangers human health. According to statistics, there are about 170 million chronically infected people worldwide. Due to the lack of good small animal models, the research on the pathogenesis of HCV is still limited to the observation of the natural history of HCV infection and the experimental infection of chimpanzees. Chimpanzees are protected animals, which are subject to many restrictions in ethics and economy, so humanization Mice are an ideal model to replace primates for HCV infection and drug screening. [0003] In recent years, Mercer (Mercer, D.F. et al. Hepatitis C virus replication in mice with chimeric human livers. Nat. Med. 7, 927-933.2001), Tateno (Tateno, C. et al. Near completely humanized liv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/11A01K67/027C12N15/63
Inventor 邓宏魁多曙光郭玉珊宋希军
Owner PEKING UNIV
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