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
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[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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