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Method for searching for target gene of Wnt signal pathway in process of differentiating embryonic stem cells into primordial germ cells

A technology of primordial germ cells and embryonic stem cells, applied in biochemical equipment and methods, genetic engineering, plant gene improvement, etc., can solve problems such as shortage, and achieve the effects of wide application, clear thinking, and simple and easy methods

Inactive Publication Date: 2019-09-24
YANGZHOU UNIV
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Problems solved by technology

[0004] In order to overcome the current defect of lacking a target gene for screening the Wnt signaling pathway, the present invention provides a method for searching for the target gene of the Wnt signaling pathway in the differentiation process of embryonic stem cells to primordial germ cells. The method is simple and feasible, and the operation is feasible Higher, providing a basis for studying how Wnt signaling regulates the formation of PGCs through target genes

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  • Method for searching for target gene of Wnt signal pathway in process of differentiating embryonic stem cells into primordial germ cells
  • Method for searching for target gene of Wnt signal pathway in process of differentiating embryonic stem cells into primordial germ cells
  • Method for searching for target gene of Wnt signal pathway in process of differentiating embryonic stem cells into primordial germ cells

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preparation example Construction

[0067] In the present invention, the preparation method of the Wnt overexpression vector preferably includes the following steps: designing primers according to the Wnt5A gene sequence, and performing PCR amplification using the reproductive organ cDNA of the animal to be tested as a template to obtain a Wnt fragment. The amplified Wnt fragment is connected to a plasmid vector to obtain a Wnt overexpression vector. In the present invention, the PCR amplification conditions preferably include: 98°C for 3 minutes; 35 cycles of 98°C for 25s, 64°C for 30s, and 72°C for 1min; 72°C for 7min. In the present invention, the PCR amplification system preferably includes: 1 μL of template, 1 μL of upstream primer, 1 μL of downstream primer, 7 μL of water, and 10 μL of amplification enzyme.

[0068] In the present invention, the preparation method of the β-Catenin overexpression vector preferably includes the following steps: design primers according to the gene sequence of β-Catenin, and ...

Embodiment 1

[0105] 1. Prediction of Wnt5A / β-Catenin / TCF7L2 signaling target genes

[0106] Due to the lack of poultry database, online prediction software: http: / / gtrd.biouml.org / bioumlweb / was used to predict the target genes bound by Wnt signaling transcription factor TCF7L2 in different species (Human, Rat, Mouse). The result is as figure 1 As shown, a total of 15374 target genes were enriched in Mouse, a total of 15727 target genes were enriched in Human, a total of 15652 target genes were enriched in Rat, among which there were 9218 common target genes in Mouse_vs_Human, 8714 common target genes in Mouse_vs_Rat8, and 10016 in Human_vs_Rat There are 7473 common target genes among the three species. In order to screen highly conserved target genes, this study selected 7473 target genes shared by the three species for further analysis. Use the online database DAVID: https: / / david.ncifcrf.gov / and KOBAS: http: / / kobas.cbi.pku.edu.cn / anno_iden.php to perform GO (Gene Ontology, Geneontolo...

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Abstract

The invention provides a method for searching for a target gene of a Wnt signal pathway in a process of differentiating embryonic stem cells into primordial germ cells and relates to the technical field of biology. According to the method, firstly, target gene prediction is carried out on a key transcription factor TCF7L2 of the Wnt signal pathway, GO function annotation is carried out on the predicted target gene, and a conservative target gene which is involved in germ cell development and stem cell differentiation is screened out as a to-be-selected target gene. In-vivo / in-vitro experiments are carried out to verify the regulation and control effect of a Wnt signal on the transcription level of the to-be-selected target gene, and when a transcription factor binding site in a promoter of the to-be-selected target gene is lost, whether or not the Wnt signal has a regulation effect on the transcription level of the to-be-selected target gene is detected; the binding effect of a beta-Catenin / transcription factor complex and the to-be-selected target gene is verified through ChIP-qPCR in order to determine whether or not the to-be-selected target gene has a targeting response effect on the Wnt signal. The method is simple and feasible.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to a method for finding the target gene of the Wnt signaling pathway in the differentiation process of embryonic stem cells to primordial germ cells. Background technique [0002] The specialization of ESCs (embryonic stem cells) to PGCs (primordial germ cells) is the first key step in germ cell generation. Although the mechanisms of PGCs specialization are different among different species, they all combine the inhibition of somatic developmental programs, multiple The initiation of competent cellular programs and the complex dynamic changes in three aspects of genome-wide epigenetic reprogramming. At present, the research on the former two is relatively thorough, and more and more evidences show that the inhibition of somatic cells and the initiation of pluripotent cells are both subject to the molecular regulation of some of the same gene expression, including cytokines, RN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N5/10C12Q1/686
CPCC12N15/86C12N2740/15043C12Q1/686
Inventor 李碧春张晨左其生王曼金晶李婷婷张亚妮孙红艳
Owner YANGZHOU UNIV
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