Efficient Delivery of DNA and Morpholinos into Mouse Preimplantation Embryos by Electroporation 英文参考文献.docVIP

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Efficient Delivery of DNA and Morpholinos into Mouse Preimplantation Embryos by Electroporation 英文参考文献.doc

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Efficient Delivery of DNA and Morpholinos into Mouse Preimplantation Embryos by Electroporation 英文参考文献

EfficientDeliveryofDNAandMorpholinosintoMouse PreimplantationEmbryosbyElectroporation HuiPeng,YongyanWu,YongZhang* CollegeofVeterinaryMedicine,NorthwestAFUniversity,KeyLaboratoryofAnimalBiotechnology,MinistryofAgriculture,Yangling,Shaanxi,People’sRepublicofChina Abstract Mouse preimplantation development is characterized by three major transitions and two lineage segregations. Each transition or lineage segregation entails pronounced changes in the pattern of gene expression. Thus, research into the function of genes with obvious changes in expression pattern will shed light on the molecular basis of preimplantation development. We have described a simplified and effective method–electroporation–of introducing plasmid DNA and morpholinosintomousepreimplantationembryosandverifiedeffectivenessofthisapproachbytestingtheprocedureon theendogenousgeneOct4.Beforeelectroporation,thezonapellucida wasweakenedbythetreatmentofacid Tyrode’s solution.Thenweoptimizedtheparameterssuchasvoltage,pulseduration,numberofpulsesandrepeats,andapplied theseparameterstosubsequentexperiments.Comparedwiththecontrolgroups,thenumberofapoptoticcellsandthe expressionandlocalizationofOCT3/4orCDX2wasnotsignificantlychangedinblastocystsdevelopedfrom1-cellembryos, which were electroporated with pIRES2-AcGFP1-Nuc eukaryotic expression vector or mismatched morpholino oligonucleotides.Furthermore,electroporatedplasmidDNAandmorpholinostargetingtheendogenousgeneOct4were able to sharply down regulate expression of OCT4 protein and actually cause expected phenotypes in mouse preimplantation embryos. In conclusion, plasmid DNA and morpholinos could be efficient delivered into mouse preimplantation embryos by electroporation and exert their functions, and normal development of preimplantation embryoswasnotaffected. Citation: Peng H,WuY,ZhangY(2012) EfficientDelivery ofDNA andMorpholinosintoMousePreimplantationEmbryos byElectroporation.PLoSONE 7(8): e43748.doi:10.1371/journal.pone.0043748 Ed