The Functional Study Of Wdr82 In Mouse Embryonic Stem Cell

Objective:1.To explore the role of miR-22 in the maintenance of pluripotency in mouse embryonic stem cells.2.To explore the function of Wdr82 in controlling mouse embryonic stem cells self-renewal and differentiation.Methods:1.This study aims to examine the regulatory roles of miR-22 in mouse embryonic stem cell(m ESC)through generating miR-22 knockout m ESC with CRISPR/Cas9 technique.First,point mutation and overlap PCR were performed to construct neomycin resistant sg RNA expression plasmids.Then,sg RNA expression plasmids targeting miR-22 were electroporated into m ESC with stable Cas9 expression.After neomycin selection and PCR genotyping,we identified miR-22 homozygous knockout m ESCs.At last,the function of miR-22 in m ESC was analyzed.2.In order to explore the function of Wdr82 in the maintenance of pluripotency and lineage differentiation in m ESC,we generated the m ESC with Wdr82 conditional knockout or Wdr82 inducible overexpression.First,we combined CRISPR/Cas9 and Cre-Lox P system to generate Wdr82 conditional knockout clones rapidly and effectively.Second,Wdr82 homozygous deletion was induced by 4-OHT treatment and pluripotency markers were examined through RT-q PCR.Third,embryoid experiment and RT-q PCR assays were performed to detect the the expression of ectoderm,mesoderm,endoderm and trophectoderm markers.Finally,Wdr82 overexpression line was constructed and then rescue experiment was carried out.Results:1.Loss of miR-22 has no obvious effect on the morphology of m ESC as well as the expression of pluripotent markers including Oct4,Sox2 and Nanog.2.Compared with the controls,the expression of pluripotent markers as well as cell morphology were not affected upon Wdr82 deletion.3.During embryoid body(EB)formation,the endoderm markers(Gata6,Sox17)in Wdr82-deficient ES cells were highly expressed in advance,while the expression of mesoderm and ectoderm markers(T,Mixl1,Fgf5)were obviously decreased.Besides,the expression of neuroectodermal markers didn't change significantly.Then we overexpressed Wdr82 in Wdr82-deficient ES cells and found the differentiation defects to three germ layers were restored to normal levels.Conclusions:1.In this study,CRISPR/Cas9 technology was used to construct miR-22 knockout mouse ES cells.miR-22 may be not required for the maintenance of embryonic stem cell,while the roles of miR-22 in controlling m ESC differentiation and cell fate decision need further identification.2.We used the novel CRISPR/Cas9 based conditional knockout technology to construct Wdr82 deficient ES cells and found Wdr82 deletion exibits no obvious defects on cell morphology and pluripotent markers expression.However,Wdr82 deletion results in the suppression of ectoderm and mesoderm differentiation whilepromoting endoderm differentiation.Besides,the expression of neuroectodermal markers was not affected significantly.At the same time,Wdr82 overexpression could rescue the differentiation defects caused by Wdr82 deletion.The above results indicate that Wdr82 plays an important role in controlling the differentiation of three germ layers.However,further efforts are still needed to explore the molecular mechanism of Wdr82 in regulating ES cell differentiation.