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Method for promoting in-vitro embryonic development by regulating metabolism

An embryo development and embryo technology, applied in the field of promoting embryo development in vitro, can solve the problems of metabolic gene expression, changes in the dynamic characteristics of epigenetic states, etc., and achieve the effect of promoting embryo development.

Active Publication Date: 2021-09-24
ZHEJIANG UNIV +1
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  • Abstract
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Problems solved by technology

Therefore, the changes in the expression of metabolic genes, epigenetic state and their dynamic characteristics during early mouse embryonic development are still not revealed.

Method used

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  • Method for promoting in-vitro embryonic development by regulating metabolism
  • Method for promoting in-vitro embryonic development by regulating metabolism
  • Method for promoting in-vitro embryonic development by regulating metabolism

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Experimental program
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preparation example Construction

[0060] 3. Preparation of dual reporter gene ES cell lines

[0061] E14 stably transfected reporter gene 2C::tdTomato was used to label two-cell like cells (2-cell like cells, 2CLC). Transfected Nanog::GFP labeled naive cells. After cell transfection, the 2C::tdTomato plasmid was transfected with lip2000 for 7 days and then selected with 150 μg / ml hygromycin for 48 hours. E14 cells containing dual reporter genes 2C::tdTomato and GFP were cultured in LIF / Serum medium.

[0062] 4. Sorting a small number of cells by flow cytometry

[0063] Optimizing the metabolomics of a small number of cells: ES cells (2C-like: tdTomato positive, ES: GFP positive) containing a dual reporter system were digested with trypsin for 5 minutes, the serum-containing medium was stopped, and then placed in a 37°C incubator for 30 minutes. The MEFs were allowed to attach and thus separated from the ES cells. The ES cells suspended in the supernatant were collected, washed once with 0.2% BSA / PBS, and f...

Embodiment 1

[0168] Example 1 Metabolomics analysis shows that the mitochondrial TCA cycle is significantly enhanced during the transition from 2-cell to blastocyst

[0169] To understand the dynamic metabolic remodeling during preimplantation embryo development, we employ a mass spectrometry-based few-cell metabolomics approach to directly detect the abundance of various metabolites in embryos. We collected 100 embryos at the 2-cell stage and 100 embryos at the blastocyst stage, representing the totipotent state, that is, when the zygotic genome is activated, and the pluripotent state, that is, when ES cells are generated, for targeted metabolomics analysis. Each condition contained three replicates of biological samples ( figure 1 in A). A total of 113 metabolites were detected (Table 1). Compared with the quality control, the experimental group (including 2c: embryos at 2-cell stage; BC: embryos at blastocyst stage; GFP: ES cells; 2c-like: 2-cell-like cells; QC: blank (group containin...

Embodiment 2

[0177] Interrelationship between embodiment 2α-KG and 2-HG

[0178] To understand whether metabolites are directly involved in gene regulation and embryonic development, we chose α-KG and 2-HG, which have established roles in epigenetic regulation, as research objects. 2-HG has two enantiomers, D-2-HG and L-2-HG, and it was recently found that the L-form 2-HG was found under certain physiological conditions. The very high levels of 2-HG that occur when genome-wide epigenetic reprogramming occurs in early embryos led us to investigate further the isoforms and absolute concentration. We use isotope-labeled internal standard to distinguish the type of 2-HG ( Figure 4 in A). Unexpectedly, we found that L-2-HG ( Figure 4 In B) instead of D-2-HG, there is a high concentration in MII oocytes at the zygotic stage and 2-cell embryos, and gradually decreases as the embryonic development progresses ( Figure 4 C in and Figure 4 in D). The absolute concentration of α-KG in blast...

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Abstract

The invention provides a method for promoting in-vitro embryonic development by regulating metabolism, and relates to the technical field of biology. The inventor researches and finds that in the in-vitro embryo culture process, addition of L-2-hydroxyglutaric acid can hinder erasing of histone methylation and embryo development, and reduction of the content of L-2-hydroxyglutaric acid in an embryo can promote erasing of histone methylation and embryo development. Therefore, the invention provides the method for regulating in-vitro embryonic development of mammals, which comprises the step of promoting embryonic development by reducing the content of L-2-hydroxyglutaric acid in embryos, or inhibiting embryonic development by increasing the content of L-2-hydroxyglutaric acid in embryos. By adopting the method provided by the invention, regulation and control on in-vitro embryonic development can be effectively realized, and a new view is provided for a molecular regulation and control mechanism in an early embryonic development process.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for promoting embryonic development in vitro by regulating metabolism. Background technique [0002] Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced reprogrammed stem cells (induced pluripotent stem cells, iPSCs), are important in regenerative medicine because of their ability to differentiate into a variety of adult cells. an important tool. The signaling pathways, gene expression regulation, and epigenetic regulation related to pluripotent stem cells have been relatively in-depth studies, while the research on pluripotent stem cell metabolism has just begun to reveal the role of metabolism in connecting the extracellular environment, the metabolic network in the cytoplasm, and the nucleus. important role in the regulation of gene expression. However, how these regulations affect the acquisition, maintenance and exit of stemness in pluripo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/53C12N15/87
CPCC12N5/0604C12N9/0006C12N15/87C12Y101/99002C12N2510/00
Inventor 张进胡泽平赵静姚珂徐雨雁张玲
Owner ZHEJIANG UNIV
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