dlk1dio3基因簇长非编码rna在小鼠早期胚胎的表达及甲基化分析word格式论文.docxVIP

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dlk1dio3基因簇长非编码rna在小鼠早期胚胎的表达及甲基化分析word格式论文.docx

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dlk1dio3基因簇长非编码rna在小鼠早期胚胎的表达及甲基化分析word格式论文

AbstractRecentyears,longnon-codingRNA(lncRNA)inDlk1-Dio3geneclusterhavegainedwidespreadattentionbecauseoftheircloselyrelationshipwithinducedpluripotentstem cell(iPS).Thisstudyfocusedon the mainlncRNAs(Gtl2,RianandMirg)inDlk1-Dio3genecluster,andanalyzedtheirsspatiotemporalexpressionpatternsbyinsituhybridizationandfluorescenceinsituhybridization(FISH).andthemethylationstatusoftwoimportantdifferentiallymethylatedregions(IG-DMR,Gtl2-DMR)weredetectedbybisulfitegenomicsequencingduringmousepreimplantationdevelopment.Additionally,varietyexpressionoflncRNAsandmethylationdynamicofDMRsinTM3treatedwithmethyltransferaseinhibitorwerealso analyzed.Firstly aprotocol ofwhole in situ hybridization basedonporeplateand dropwasestablishedinthisstudy.InsituhybridizationindicatedthattheselncRNAsonlyexpressed in the morula and blastocyst, while not in 8-cell stage and before.Interestingly, the expressionoflncRNAs ishigher inblastocyststage thanthatinmorula,whichinaccordancewiththeresultsofreal-timeRT-PCR.Moreimportantly,wefoundtheexpressionsignalsoftheselncRNAswereconcentratedintheinnercell mass (ICM) of the blastocyst, but almost not in trophectoderm (TE). FISHillustratedthattheselncRNAs’signalmainly surrounding cellnucleusinblastocyst.Asanalyzedbybisulfitesequencing analysis,from2-cell tomorulastage,IG-DMRstill maintaineditsdifferentiallymethylatedstateestablishedinthegermline.Thatis maternal non-methylated and paternal methylated. Gtl2-DMR showed lowmethylatedstateonbothmaternalandpaternalstrandsthroughoutthepreimplantationdevelopment,andappearednoobviouschanges.Whatsmore,theexpressionoftheselncRNAswasnotregulatedbyDNAmethylationinTM3.ThisisthefirststudytoclarifytheexpressionpatternsoflncRNAsinDlk1-Dio3geneclusterduringthepreimplantationdevelopment.TheexpressionoflncRNAsatmorulaandblastocyststage,andintensesignalsfromICM ofblastocyststronglyindicatedthepossibleassociationbetweentheselncRNAsand ESCs. ThemethylatedstatesofDMRsdidnotchangeduringthepreimplantationdevelopment.Itseem