Construction method and application of HBeAg transgenic mouse model

A technology of transgenic mice and construction methods, applied in the field of biomedicine, can solve problems such as constraints, low construction efficiency, and failure to achieve liver-specific expression of HBeAg

Inactive Publication Date: 2018-08-21
XI AN JIAOTONG UNIV
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Problems solved by technology

Although some scholars previously constructed HBeAg transgenic mice (Milich DR, et al. 1990.) and HBc/HBeAg transgenic mice confirmed that HBeAg can induce stronger T cell immune tolerance than HBcg (Liu Hong, 2003), but now Some models f

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  • Construction method and application of HBeAg transgenic mouse model
  • Construction method and application of HBeAg transgenic mouse model
  • Construction method and application of HBeAg transgenic mouse model

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Embodiment Construction

[0040] The present invention will be further described in detail below in conjunction with specific embodiments, which are explanations of the present invention rather than limitations.

[0041] The present invention is achieved through the following technical solutions:

[0042] 1. Construction of recombinant R26-e(Alb-HBeAg)1 targeting vector:

[0043] (1) First use conventional methods to clone the HBeAg gene of HBV to obtain the HBeAg gene, and then use CRISPR / Cas9 technology (Mali P, et al.2013; Cong L, et al.2013; Wang H, et al.2013; Shen B, etal.2013.), the HBeAg gene was inserted into the corresponding expression frame at the Rosa26 gene locus by means of homologous recombination, such as figure 1 shown.

[0044] 1) Overexpression target gene name: HBeAg

[0045] 2) Insertion site gene name (Ensembl): Gt(ROSA)26Sor (ENSMUSG00000086429), abbreviation: Rosa26.

[0046] 3) Ensembl website link of the target gene at the insertion site:

[0047] http: / / asia.ensembl.org...

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Abstract

The invention provides a construction method and an application of an HBeAg transgenic mouse model. The method comprises the following steps: CRISPR/Cas9 technology is adopted, an HBeAg gene is inserted into a pliver-HBeAg expression cassette at the Rosa26 gene locus in a fixed-point manner through homologous recombination, and a recombinant R26-e(Alb-HBeAg)1 targeting vector is constructed with an In-Fusion cloning method and contains a 3.3kb 5' homologous arm, pliver-HBeAg and a 3.3kb 3' homologous arm; Cas9mRNA, gRNA and the recombinant R26-e(Alb-HBeAg)1 targeting vector are micro-injectedinto a zygote of a C57BL/6J mouse, then the zygote is transplanted into the uterus of a C57BL/6J female mouse, and an HBeAg transgenic mouse is obtained through propagation and purification step by step. The transgenic mouse can specifically express HBeAg in liver, thereby being capable of applying to experimental animal models for researching HBeAg infection mechanism and evaluating treatment effects of therapeutic drugs and vaccines for chronic hepatitis B.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to the preparation of a medical transgenic mouse model animal, in particular to a construction method and application of an HBeAg transgenic mouse model. Background technique [0002] Hepatitis B virus (HBV) is a DNA virus belonging to the family Hepadnaviridae. Acute and chronic hepatitis B and secondary lesions (cirrhosis, hepatocellular carcinoma, etc.) caused by HBV infection seriously endanger human health. Since HBV was discovered in 1967, about 2 billion people worldwide have been infected with HBV, and 240 million of them are HBV carriers. Therefore, it is of great scientific value and great social and economic significance to study the mechanism of HBV infection and explore strategies for the prevention and treatment of hepatitis B. [0003] However, due to the strict species restriction and hepatotropic characteristics of HBV, and in addition to humans, although chimpanz...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N15/51A01K67/027A61K49/00
CPCA01K67/0275A01K2217/072A01K2227/105A01K2267/0337A61K49/0008C07K14/005C12N9/22C12N15/8509C12N15/907C12N2730/10122C12N2800/107
Inventor 杨军张明娟郭睿黄小钟李宗芳
Owner XI AN JIAOTONG UNIV
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