Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation.
Conditions for the electroporation of mouse oocytes and preimplantation embryos have been optimised by following the incorporation of rhodamine labeled dextran. This procedure includes a step to weaken but not remove the zona pellucida that helps achieve good survival. This approach has been applied to introduce double-stranded RNA for c-mos into oocytes and green fluorescent protein (GFP) into transgenic GFP-expressing embryos at the 1- and 4-cell stages. In both cases we were able to observe sequence-specific interference with the expression of the target gene--a failure of oocytes to arrest at metaphase II and a loss in the green fluorescence of embryos by the morula or blastocyst stages. These effects could be observed in multiple oocytes or embryos allowed to develop together following electroporation.
Grabarek
Joanna B
JB
Cyclacel Ltd., Polgen Division, Babraham Bioincubator, Babraham Cambridge, United Kingdom.
Plusa
Berenika
B
Glover
David M
DM
Zernicka-Goetz
Magdalena
M
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D017931:DNA Primers; D018274:Electroporation / Q000379:methods; D016258:Genes, mos; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D009865:Oocytes / Q000502:physiology*; D017231:Proto-Oncogene Proteins c-mos / Q000235:genetics*; D012330:RNA, Double-Stranded / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results; D015044:Zona Pellucida / Q000502:physiology*
D017931:DNA Primers; D008164:Luminescent Proteins; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D017231:Proto-Oncogene Proteins c-mos