| Embryonic stem cells are pluripotent cells derived from mammalian early embryos,which can proliferate and differentiate indefinitely in vitro and have the potential to develop into intact individuals.Embryonic stem cells express transcription factors such as Oct4,Sox2 and Nanog,which form a pluripotent regulatory network and maintain the pluripotentcy of ESCs by regulating the expression level of each other.Mitochondrial metabolism plays a very important role in determining the fate of stem cells.Previous studies have shown that the maintenance of mitochondrial homeostasis plays a key role in maintaining the self-renewal ability and pluripotency of stem cells,but the specific regulatory mechanisms need to be further clarified.In normal somatic cells,mitochondria produce a small amount of ATP by glycolysis in anoxic environment,and ATP is mainly produced by oxidative phosphorylation under aerobic conditions.The embryonic stem cell mitochondrial energy supply and specific mechanisms have not yet reached a clear conclusion.Complex ? is at the core of the electron transport chain,and it continues to transfer electrons from complex ? and complex ? through coenzyme Q to cytochrome c,while pumping protons out of the mitochondrial inner membrane to form a membrane potential.As an important assembly factor in mitochondrial complex ?,TTC19 plays a key role in the formation of complex ? dimer and in the regulation of mitochondrial electron transport and oxidative phosphorylation.We used mouse embryonic stem cells as the research object,and selected the mitochondrial complex ? assembly factor TTC19 as a target to study the effect of oxidative phosphorylation defects on the maintenance of embryonic stem cell pluripotency.In this study,we first preliminarily analyzed the function of mitochondria in mouse embryonic stem cells and embryonic fibroblasts.Compared with somatic cells,the oxidative phosphorylation of embryonic stem cells is low-coupling with ATP production,but it also has complete mitochondrial function.Using the CRISPR/Cas9 gene editing technology,the gRNA was designed on the website by using the exon of Ttc19 gene as the target sequence,and the pX330-gRNA-Cas9 plasmid was constructed,and the plasmid was transfected into mouse ESCs by nuclear transfection..After detecting the efficiency of the cleavage,we selected the plasmid corresponding to the most efficient gRNA to construct Ttc19 knockout mouse ES cell lines.After selecting the monoclonal,the culture is expanded.When the number of cells is sufficient,some cells are collected to extract the genomic DNA,and the target sequence to be detected and the DNA fragment of about 700 bp are amplified.After TA cloning,a single colony is picked and sent for sequencing.According to the sequencing results,two cell lines corresponding to the edited results were selected,that is,Ttc19 knockout cell lines for our needs,and the karyotype test showed normal diploid karyotype.Compared with wild-type ESCs in energy metabolism,Ttc19 knockout ESCs mitochondrial oxidative phosphorylation function was significantly inhibited.At the same time,self-renewal ability of Ttc19 knockout ES cell lines was detected,and the expression of pluripotency genes at transcriptional level and protein level was detected.We found that their pluripotency was also significantly lower than that of wild-type ESCs.The results showed that deletion of Ttc19 gene resulted in a defect in the oxidative respiratory chain of embryonic stem cells,which reduced their pluripotency.In this study,ES cell lines with Ttc19 gene knockout were successfully constructed,which provided an important cell model for further study of the mechanism of mitochondrial function and stem cell fate regulation. |
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