Constructing method of 1.3 copy C gene type HBV transgenic mice

A technology of transgenic mice and construction methods, applied in the field of animal model construction, can solve the problems of less research on C-type transgenic mice, and achieve the effects of improving transgenic efficiency, abundant research methods and high efficiency

Inactive Publication Date: 2014-07-30
中国人民解放军第四五八医院
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  • Abstract
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  • Application Information

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Problems solved by technology

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  • Constructing method of 1.3 copy C gene type HBV transgenic mice
  • Constructing method of 1.3 copy C gene type HBV transgenic mice
  • Constructing method of 1.3 copy C gene type HBV transgenic mice

Examples

Experimental program
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experiment example 1

[0061] Experimental Example 1 Construction of 1.3 copy C genotype HBV transgenic mice

[0062] 1. Establishment of embryonic stem cell (ES cell) line

[0063] (1) Take C57BL / 6J female pregnant mice 3.5 days of pregnancy, and kill them by breaking their necks.

[0064] (2) Use 75% alcohol to disinfect the pregnant mice killed by S11, and spray the whole body as much as possible.

[0065] (3) Open the abdominal cavity of the pregnant mouse, repair and remove the fat and mesangium, cut the bilateral uterus from the root of the fallopian tube and the bifurcation of the lower end of the uterus, and cut the bicornuate uterus at the vagina.

[0066] (4) The syringe sucks M2 (purchased from American Sigma company) as the embryo preparation solution. Before using the syringe needle, cut off the tip of the needle and sterilize it; insert the syringe from one end of the uterus into the uterine cavity 2~3mm, clamp it, Blow in the embryo flushing fluid, then insert the syringe from the other end o...

experiment example 2 Embodiment 1

[0093] Experimental Example 2 Example 1 Preparation of 1.3 copies of Type C HBV transgenic mice

[0094] 1. Statistics of mouse mosaic rate

[0095] (1) Judging the chimeric rate of mice based on the color of the mouse's coat: Since the ES cells are derived from black-haired C57BL / 6J mice, and the blastocysts are derived from white-haired KM mice, the judgement is based on the percentage of black hair in the born mice Mice chimerism rate.

[0096] (2) Combine the obtained chimeric mice (F0 generation) and normal KM mice in a cage. If the obtained F1 generation mouse is a chimeric mouse, it is proved that the F0 generation chimeric mouse has reproductive chimerism and can be inherited to the next generation.

[0097] 2. ELISA detects hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in serum and urine of transgenic mice. The detection methods are as follows:

[0098] (1) Detection of hepatitis B surface antigen (HBsAg) (ELISA method)

[0099] Use the hepatitis B vir...

experiment example 3 Embodiment 1

[0122] Experimental Example 3 Example 1 Preparation of 1.3 copies of Type C HBV transgenic mice Passage identification

[0123] 1. Combine the chimeric mice (F0 generation) prepared in the example and normal KM mice to obtain 32 F1 generation mice.

[0124] 2. The statistics of mouse mosaic rate and the method of ELISA to detect hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the serum and urine of transgenic mice are the same as in Example 2.

[0125] 3. Identification results

[0126] (1) Five of the 32 F1 generation mice are positive chimeric mice, and the positive rate of chimeric mice is 15.6%, indicating that the method of the present invention constructs chimeric mice that can be passed down.

[0127] (2) The 5 F1 chimeric mice were numbered B1~B5.

[0128] The results of ELISA detection of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) showed that HBsAg and HBeAg in serum and urine of B1~B5 mice were positive. The measured OD valu...

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Abstract

The invention provides a constructing method of 1.3 copy C gene type HBV transgenic mice. Through blastocyst injection of ES cells, the method prepares targeted integration 1.3 copy C gene type HBV (Hepatitis B virus) transgenic mice. First, targeted integration1.3 copy C type HBV expression vectors are constructed, and then an electrical transduction transformation method is employed to transform recombinant plasmids into ES cells; after the and cultivation, targeted integration recombinant ES cells are obtained; then the targeted integration recombinant ES cells are digested into single cells and injected into blastocysts; and then the blastocysts are transplanted to uteruses of pseudopregnant female mice. The method sets up the 1.3 copy C type HBV transgenic mice for the first time home and abroad, is specifically applied to the field of hepatitis B related researches, and provides a more ideal animal model for the researches on prevention and treatment of hepatitis B in China.

Description

technical field [0001] The invention relates to the field of animal model construction. More specifically, it relates to a method for constructing HBV transgenic mice with 1.3 copies of genotype C. Background technique [0002] Hepatitis B virus (HBV) infection is a major problem affecting human health worldwide. Chronic progressive liver inflammatory lesions caused by HBV infection often develop into cirrhosis, liver cancer and eventually death. According to the report of the World Health Organization, about 2 billion people in the world have been infected with HBV, of which 350 million people are chronically infected with HBV, and the number of deaths from this is about 1 million to 2 million every year. my country has always been a high-incidence area of ​​HBV infection. The latest data show that there are about 93 million chronic HBV infections in my country, of which about 20 million are chronic hepatitis B patients, accounting for more than 1 / 5 of chronic HBV infecti...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N5/0735A01K67/027
Inventor 刘光泽陈媚娟李秀梅谢勇张振伟孔祥平
Owner 中国人民解放军第四五八医院
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