Construction Of DUSP9 Knockout Mouse Embryonic Stem Cell Line By CRISPR-Cas9 System

Mouse Embryonic Stem Cells(m ESCs)are derived from the inner cell mass of preimplantation blastocysts and have unlimited self-renewal ability.Martin Evans et al.established firstly a mouse ESCs cell line using feeder cells and serum systems in 1981.The research on culture system of m ESCs has attracted more and more attention in vitro.In 2008,Ying et al derived mouse na?ve state ESCs from blastocysts in 2i/LIF medium which contains MEK inhibitor PD0325901,GSK3 inhibitor CHIR99021(2i)and leukemia inhibitory factor(LIF).In 2018,Bao et al.successfully established Advanced Stem Cells(ASCs)in chemical defined medium with adding Activin A,BMP4,CHIR99021 and LIF(ABC/L-medium).Transcriptome data of ASCs and m ESCs showed that Dusp9 expression in ASCs was significantly up-regulated.Based on this discovery,we constructed a Dusp9 knockout ASCs cell line using the CRISPRCas9 system and identified the pluripotency,DNA methylation and developmental potential of the Dusp9 knockout ASCs.Furthermore,we used real-time quantitative PCR and immunofluorescence staining to detect the expression of genes and proteins related to DNA methylation and glycolipid metabolism in Dusp9 knockout ASCs.The result as follows:1.Established Dusp9 knockout ASCsFirstly,we constructed the PX-459 vector contained Dusp9-sg RNA and transfect this recombinant vector into ASCs via Lipofectamine? 2000 Transfection Reagent.The Dusp9 knockout ASCs was successfully verified by PCR sequencing,q PCR and WB.2.Transcriptome analysis of Dusp9 knockout embryonic stem cell lineTranscriptome analysis showed that Dusp9 expression difference influence the maintenance of pluripotency of embryonic stem cells and the expression of related regulatory genes.To further GSEA analysis,we found there are also significant differences in signal pathways,such as m TOR and fatty acid metabolism in Dusp9 knockout embryonic stem cell line.Finally,the expression of genes related to glucose and lipid metabolism in Dusp9 knockout cells were verified by q PCR.3.Pluripotency,DNA methylation and developmental potential of Dusp9 knockout embryonic stem cellsFirst,the pluripotency of Dusp9 knockout embryonic stem cells was detected by AP staining,q PCR and immunofluorescence staining.The results show that the Dusp9 knockout embryonic stem cell line has the characteristics of stem cell pluripotency with AP positive staining,and also expressed Oct4 and Sox2.Secondly,it was verified that the deletion of Dusp9 caused the decrease of Dnmt3 B expression on the m RNA and protein levels.At the same time,in vitro differentiation experiments in vitro showed that the expression level of the three germ layer marker genes in Dusp9 knockout cell line was significantly higher than that of the control group,indicating it has a higher potential for three germ layer differentiation.Notably,the results of chimera experiments show that Dusp9 knockout cells can contribute to the fetus and yolk sac tissue,The results revealed Dusp9 knockout cells have developmental potency.In summary,we have successfully obtained Dusp9 knockout ASCs cell line using CRISPR-Cas9 technology.We found the loss of Dusp9 affected the expression of genes related to DNA methylation,glucose and lipid metabolism.Furthermore,the Dusp9 knockout embryonic stem cells have development pluripotency,and contributed to post-implantation embryos and yolk sac by chimera production.