title: Efficient delivery of DNA and morpholinos into mouse preimplantation embryos by electroporation.
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 morpholinos into mouse preimplantation embryos and verified effectiveness of this approach by testing the procedure on the endogenous gene Oct4. Before electroporation, the zona pellucida was weakened by the treatment of acid Tyrode's solution. Then we optimized the parameters such as voltage, pulse duration, number of pulses and repeats, and applied these parameters to subsequent experiments. Compared with the control groups, the number of apoptotic cells and the expression and localization of OCT3/4 or CDX2 was not significantly changed in blastocysts developed from 1-cell embryos, which were electroporated with pIRES2-AcGFP1-Nuc eukaryotic expression vector or mismatched morpholino oligonucleotides. Furthermore, electroporated plasmid DNA and morpholinos targeting the endogenous gene Oct4 were 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 embryos was not affected.
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Peng
authors.forename: Yongyan
authors.initials: Y
authors.lastname: Wu
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D005260:Female; D055785:Gene Knockdown Techniques; D051379:Mice; D060172:Morpholinos / Q000235:genetics / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000172:deficiency / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D015044:Zona Pellucida / Q000378:metabolism
chemical_list: D060172:Morpholinos; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D034741:RNA, Small Interfering; D004247:DNA