The Effect Of Maternal Factor Patl2 On Somatic Cell Reprogramming

Since the Shinya Yamanaka laboratory of Kyoto University in Japan found that Oct4,Sox2,Klf4 and c-Myc four factors can be used to induce somatic cells into induced pluripotent stem cell(iPSC)in 2006,somatic cell reprogramming techniques have been greatly developed.But there are still various problems,such as low efficiency,long induction time and poor safety.Somatic cell nuclear transfer and early embryonic development studies have shown that maternal factors specifically present in the oocyte cytoplasm initiate early embryonic development,activate genome transcription and synthesize new genes,and finally produce a cascade reaction to maintain the continued the development of embryos.These factors can improve the efficiency of early embryonic development and reprogramming.The paternal factors located in the male pronucleus are also important factors in early embryonic development and nuclear transfer.Studies have shown that the fusion of an additional male pronucleus during nuclear transfer can significantly improve the efficienc of nuclear transfer.These studies further confirmed that key factors present in the oocyte cytoplasm and male pronucleus can promote reprogramming.The purpose of this study was to screen maternal or paternal factors that can promote reprogramming,and to identify the iP cell lines produced,and then to explore the mechanism of reprogramming.First,based on the reported early embryo proteome data,we selected 6 maternal and paternal factors,Patl2,Grn,Pdha1,Faf1,Dhodh and Lman1,as candidate genes,and constructed Dox-induced over-expression vectors of 6 genes.To test the effects of six factors on mouse somatic cell reprogramming based on the classic four-factor induction.We used the fibroblasts of Oct4-GFP/Rosa26-M2rtTA transgenic mice as the starting cells for screening,and found that in the six screening factors,over-expression of maternal factor Patl2 or paternal factor Grn,Faf1 can increase the induction efficiency of somatic cell reprogramming.Among them,over-expression of Patl2 has the most significant effect on reprogramming.After the reprogramming,samples were collected and subjected to flow cytometry.It was found that the number of Oct4-GFP~+cells produced by over-expressing Patl2 was 15 times higher than that of the control vector.The expression of the pluripotent gene Oct4 is a sign that the cells are completely reprogrammed.Addition of Patl2 allows more fibroblasts to reprogram into induced pluripotent stem cells.At the same time,the number of clones produced by over-expressing Patl2 shown by alkaline phosphatase staining(AP staining)also increased significantly,and was 12 times than that of the control group.And the over-expression of Patl2 during the reprogramming process can speed up the time of clone appearance.To further determine the effect of Patl2 on reprogramming,we selected single clones after the induction to establish the OSKM+Patl2-iPS cell line.The study found that the cell line had similar morphological characteristics as embryonic stem cells and showed green fluorescence due to the expression of the GFP-tagged multipotent gene Oct4.Next,we also verified this view at the RNA level.Through detection of the expression of pluripotent genes in the OSKM+Patl2-iPS cell line,we found that the pluripotent gene can be expressed at a high level and is significantly higher than the MEF of the starting cell.This result was also confirmed at the protein level.We selected three pluripotency marker proteins NANOG,SOX2,and SSEA1 for immunofluorescence staining of the cell lines.The selected OSKM+Patl2-iPS cell lines all showed red fluorescence and proved that the corresponding pluripotent protein expressed.In addition,we have also verified the cell lines in terms of differentiation potential.First,the karyotype test was performed on the cell line established by over-expression of Patl2to ensure normal karyotype.Afterwards,the embryonic body(EB)experiment was carried out by using the hanging drop method to simulate the embryonic environment.The experiment proved that OSKM+Patl2-iPS cell lines can all differentiate into EB in vitro.After these EB balls adhered,quantitative detection found the expression of endoderm marker genes(Gata6,Sox17),mesoderm marker genes(Nkx2.5,Brachy),ectoderm marker genes(Fgf5,Kdr),trophoblast marker genes(Eomes,Cdx2)increased,which proved that the generated embryonic body can randomly differentiated into the endoderm,mesoderm,ectoderm.In the chimerism experiment,OSKM+Patl2-iPS chimerism with Oct4-GFP green fluorescence could be produced on the reproductive crest of the receptor embryos,which proved that OSKM+Patl2-iPS was indeed totipotent.To explore Patl2 promote specific mechanism of reprogramming,for reprogramming 3days,clone(11±2 days)and a large number of clone three time points(15±2 days)RNA-seq,the cells of the sequencing results GO analysis shows that a Vector expression Patl2compared with control group showed a large genetic differences,which raised genetic difference between enrichment in transcription,amide metabolism,peptide synthesis and processing,the ribosome,organic nitrogen compound in the process of biosynthesis;The down-regulated genes were enriched in ubiquitin-protein-transferase activity,Ras GTPase binding,phosphorylation,cell adhesion,and cell cycle.Enrichment results of KEGG pathway showed that Patl2 over-expression mainly affected ribosome and oxidative phosphorylation.Compared with Vector over-expression in the control group,Patl2 up-regulated the expression of 22 genes including ribosome component protein coding genes Rpl22,Mrpl34 and Rps16.In conclusion,our study found that new maternal factors can affect reprogramming.This highly expressed factor in the oocyte cytoplasm,Patl2,can increase the efficiency of reprogramming induction.The over-expression produced iPS cell line can increase the expression of pluripotency genes,has a normal mouse karyotype,and is able to the germ layer randomly differentiates.And these may be achieved through Patl2 affecting transcription and the mTOR pathway.These provide a new idea for exploring more efficient and safe reprogramming methods.At the same time,the discovery of new factors enriches the methods and mechanisms of reprogramming and enhances the important connotation of the research on early embryo development.