Effects of Three AKT Isoform-specific Knockouts on Self-renewal and Differentiation in Mouse Embryonic Stem Cells
10.13865/j.cnki.cjbmb.2025.01.1465
- VernacularTitle:AKT三种亚型基因敲除对小鼠胚胎干细胞自我更新和分化的影响
- Author:
Qi YANG
1
;
Shuai TANG
1
;
Lin-Lin ZHANG
1
;
Wu-Yang TANG
1
;
Ao-Xiang DOU
1
;
Yu-Hang ZHANG
1
;
Pi-Shun LI
1
;
Xiao-Feng ZHENG
1
Author Information
1. 湖南农业大学动物医学院,湖南兽药工程技术研究中心,长沙 410128
- Publication Type:Journal Article
- Keywords:
mouse embryonic stem cells(MESCs);
AKT isoform;
pluripotency;
clustered regularly in-terspaced short palindromic repeats/CRISPR associated protein 9(CRISPR/Cas9);
RNA sequencing
- From:
Chinese Journal of Biochemistry and Molecular Biology
2025;41(3):426-436
- CountryChina
- Language:Chinese
-
Abstract:
AKT,also known as Protein Kinase B(PKB),plays a critical role in cell proliferation and metabolism.There are three isoforms of AKT:AKT1,AKT2,and AKT3.The effects of these isoforms on the pluripotency and differentiation of mouse embryonic stem cells(mESCs)remain unclear.This study aims to explore the impact of three AKT isoform-specific knockouts on the self-renewal and differen-tiation of mouse embryonic stem cells.Using CRISPR/Cas9 gene-editing technology,AKT isoform-spe-cific knockout cell lines were established.The phenotypic and molecular changes were analyzed through Western blotting,flow cytometry,qRT-PCR,CCK-8 assays,Alkaline Phosphatase(AP)staining,and RNA-seq.The construction of AKT isoform-specific knockout cell lines was successful.The loss of AKT1 and AKT2 inhibited the proliferation of mESCs.The knockout of any single AKT isoform did not affect the expression of pluripotency genes at both mRNA or protein levels.However,during embryoid body forma-tion,the deletion of any of the three AKT isoforms affected the mRNA expression levels of genes in all three germ layers.Transcriptome analysis showed that compared to wild-type mESCs,995,547,and 429 differentially expressed genes(|log2FC|≧1,P＜0.05)were identified inAKT1,AKT2,and AKT3 isoform-specific knockout cells,respectively.There was some overlap in the differentially expressed genes regulated by these three isoforms.In conclusion,the independent knockout of AKT isoforms does not af-fect the maintenance of pluripotency in mouse embryonic stem cells,but they are crucial for differentia-tion.The three AKT isoforms can collectively regulate gene expression while retaining their own regulato-ry specificity.This study provides a foundation for understanding the unique and overlapping roles of AKT isoforms in stem cell biology,highlighting their importance in maintaining stem cell function and differen-tiation.
