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Method for high-efficiency establishment of genetically modified animal model through haploid stem cells

A genetic modification and animal model technology, which is applied in the field of using haploid stem cells to efficiently establish genetically modified animal models, which can solve problems such as the reduction of the percentage of embryos born to live to adult mice, the lack of practicality, and the inconsistency of imprinted sperm.

Inactive Publication Date: 2015-12-02
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

The existing experimental results show that although AH-ES cells can obtain fertile SC mice, there are still some problems: first, about half of the newborn mice show abnormal development, and the individuals are obviously smaller. Second, compared with the sperm control, AH-ES cells obtained a significantly lower percentage of embryo births and percentages of surviving to adult mice; third, high-generation haploid cells and genetically modified haploid cells Ploid cell clones are difficult to obtain healthy SC mice, which means that it is not practical to use this method to construct genetically modified mice
Combined with the existing gene imprinting research results, we speculate that the main reason why AH-ES cells cannot support the normal development of embryos may be that the imprinting state of the H19 gene is inconsistent with that of sperm

Method used

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  • Method for high-efficiency establishment of genetically modified animal model through haploid stem cells
  • Method for high-efficiency establishment of genetically modified animal model through haploid stem cells
  • Method for high-efficiency establishment of genetically modified animal model through haploid stem cells

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Embodiment Construction

[0041] experimental method

[0042] 1. Preparation of Vector

[0043] 1.1 Plasmid vector

[0044] 1.1.1 Preparation of Competent Cells

[0045] (1) Streaking plate: Dip a small amount of E.coli DH5α bacterial liquid with a sterilized inoculation loop, and streak on the LB agar plate without antibiotics to gradually dilute the bacterial liquid. Place it upside down in a 37°C bacterial incubator and incubate overnight for 12-14 hours.

[0046] (2) Pick a single colony and inoculate it into 5 ml of LB medium without antibiotics. Shake overnight at 37°C and 250 rpm.

[0047] (3) Inoculate 2ml of turbid bacterial solution into 200ml LB medium without antibiotics, and incubate with shaking at 37°C for 2-3h. Measure OD every half hour 600 , to make it 0.4-0.6.

[0048] (4) The bacteria solution was ice-bathed for 20 minutes. Transfer the ice-bathed bacterial solution to a pre-cooled 50ml centrifuge tube. 4°C, 4000rpm, 10min centrifugation.

[0049] (5) Discard the supernata...

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Abstract

The invention discloses a method for high-efficiency establishment of a genetically modified animal model through haploid stem cells. Specifically, the method for high-efficiency establishment of the genetically modified animal model through haploid stem cells comprises the following steps: a) knocking out the H19 gene of male haploid embryonic stem cells through homologous recombination; b) screening and identifying the male haploid embryonic stem cells in which the H19 gene is correctly knocked out; and c) injecting the male haploid embryonic stem cells in which the H19 gene is correctly knocked out into cytoplasm of oocyte so as to produce a semi-cloned genetically modified animal. The method provided by the invention simulates the functions of sperms by establishing an H19 gene-knocked-out male haploid embryonic stem cell system so as to highly efficiently generate fertile progeny, which can then be used for implementation of novel genetic manipulation and establishment of corresponding genetically modified animal models.

Description

technical field [0001] The invention relates to a method for establishing a genetically modified animal model, in particular to a method for efficiently establishing a genetically modified animal model using haploid stem cells. Background technique [0002] Mammalian embryonic stem (ES) cells have the characteristics of self-renewal and multi-directional differentiation potential (Pluripotency). It is not only an important resource in regenerative medicine research and future clinical cell therapy, but also has become an It is an important research tool and is widely used in the construction of genetically modified animal models. [0003] The traditional method for making genetically modified animals is to use gene targeting to genetically modify diploid ES cells, and then inject specific genetically modified ES cells into the developing diploid blastocysts to generate chimeric animals and genetically modified animals into the germline 71 . This method has been widely use...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/877A01K67/027
Inventor 黄粤刘光张美丽刘语方
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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