Method for application of H3K27me3 and demethylase KDM6A/B thereof to nuclear transfer reconstructed embryo of mouse

A technology of demethylase and an application method is applied in the application field of mouse nuclear transfer and reconstructed embryos to achieve the effects of improving blastocyst rate, oocyte reprogramming ability, and blastocyst development rate.

Inactive Publication Date: 2017-10-27
INNER MONGOLIA UNIVERSITY
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Problems solved by technology

[0008] It has been reported that in mouse embryonic development and cell differentiation, KDM6A has an important regulatory effect on key genes affecting embryonic development; like KDM6A, KDM6B is also an important demethylase of H3K27me3, encoded by autosomes, in Studies on mice have shown that KDM6B is involved in the transformation of white and brown fat by regulating the activity of key fat genes; after bone injury, KDM6B can regulate the differentiation of osteoclasts and may limit the pathogenesis of osteoporosis, and is associated with There is a certain relationship between skin repair; when nerve cells are damaged, KDM6B can limit the excessive proliferation and regeneration of Schwann cells, thereby preventing the generation of neurofi

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  • Method for application of H3K27me3 and demethylase KDM6A/B thereof to nuclear transfer reconstructed embryo of mouse
  • Method for application of H3K27me3 and demethylase KDM6A/B thereof to nuclear transfer reconstructed embryo of mouse
  • Method for application of H3K27me3 and demethylase KDM6A/B thereof to nuclear transfer reconstructed embryo of mouse

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Embodiment Construction

[0088] The technical solution of the present patent will be described in further detail below in conjunction with specific embodiments.

[0089] see Figure 1-30 , an application method of H3K27me3 and its demethylase KDM6A / B in mouse nuclear transfer reconstituted embryos, comprising the following steps:

[0090] 1) Prepare experimental animals:

[0091] Use Oct4 promoter-green fluorescent transgenic mice as oocyte donor mice (OG2) for live cell imaging;

[0092] 2) Collection of MII stage oocytes:

[0093] 3) Prepare experimental reagents:

[0094] 3.1) Embryo culture medium:

[0095] 3.2) Main reagents related to immunofluorescence and western blotting:

[0096] 4% paraformaldehyde (PFA), polyethylene glycol octyl phenyl ether, phosphate buffered saline (PBS), Tween 20, DAPI dye, antibody (Oct4) (dilution ratio 1:500), antibody ( Nanog) (dilution 1:500), Antibody (SSEA1) (1:50 dilution), Antibody (Sox2) (1:500 dilution), Antibody (H3K27me3) (1:50 dilution), Antibody (...

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Abstract

The invention discloses a method for application of H3K27me3 and demethylase KDM6A/B thereof to a nuclear transfer reconstructed embryo of a mouse. With the application method, the difference between an H3K27 trimethylation mode in a parthenogenetic embryo in each period before implantation and that in a clonal embryo, and the influence of the difference in the mode on the developmental capacity of the clonal embryo are analyzed, and the method verifies that fine adjustment of KDM6A and KDM6B can improve the blastocyst development rate of the cloned embryo and the birth rate of a cloned animal.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an application method of H3K27me3 and its demethylase KDM6A / B in mouse nuclear transfer reconstituted embryos. Background technique [0002] Somatic cell nuclear transfer refers to the technique of injecting somatic cell nuclei into the enucleated oocyte cytoplasm to obtain reconstructed embryos, which eventually develop into offspring; cell reprogramming can be achieved through nuclear transfer, cell fusion, specific transcription factors and culture. However, the only reprogramming method that can induce cells to regenerate into individuals is somatic cell nuclear transfer. Other methods can only induce induction at the cellular, molecular or biochemical level, and they all have some shortcomings. Since the establishment of somatic cell nuclear transfer technology, cloning efficiency has been at a low level, only a few reconstituted embryos can develop to term birth, usually only 1...

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Application Information

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IPC IPC(8): C12N15/877C12N15/89
CPCC12N15/8775C12N15/89
Inventor 刘雪霏杨磊李光鹏
Owner INNER MONGOLIA UNIVERSITY
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