In this study,GATA1s knockout human pluripotent stem cells(hPSCs)cell line was established,and the effect of GATA1s deletion on hematopoietic differentiation in vitro was discussed.hPSCs were gene-edited to knock out GATA1s by constructing a targeting plasmid containing a recombinant arm-knock-in fragment(GATA1 exon II-VI sequence and BGH polyA sequence)-LoxP-hygromycin screening marker-LoxP-recombination arm and a gRNA plasmid containing gRNA-Cas9.The above plasmids were introduced into the cells by electroporation.After 7 days of initial screening with hygromycin,the positive clones were subjected to electroporation to make the LoxP site specificially recombined by Cre to remove the screening marker,and further cultured by single-cell cloning.Finally,the expression of GATA1 and GATA1s proteins was verified by Western blot,and the CD34+,CD43+ and CD45+ cells were increased,but GPA+,CD41a+ and CD42b+ cells were significantly decreased in the GATA1s knockout cell line.Overexpression of GATA1 through transposon system also failed to rescue the reduced GPA+,CD41a+and CD42b+cells.In this study,we established GATA1s knockout hPSCs and found that GATA1s play an important role in hematopoietic differentiation.
维普
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