title: Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA.
abstract: RNA interference (RNAi) using double-stranded RNA has been used for the systematic analysis of gene function in invertebrate organisms. Here we have explored the use of short interfering RNA (siRNA) to knock down gene expression during the development of mammalian postimplantation embryos. The developing CNS system of embryonic day 10 mouse embryos was used as a model tissue. siRNA prepared by endoribonuclease digestion (esiRNA) was injected into the lumen of the neural tube at specific regions and delivered into neuroepithelial cells by directed electroporation. Injected and electroporated embryos were grown for 1 day in whole-embryo culture and the effects of RNAi were examined. esiRNA directed against beta-galactosidase (beta-gal), coelectroporated into neuroepithelial cells together with reporter plasmids expressing GFP and beta-gal, abolished expression of beta-gal but not GFP, showing the specificity of the esiRNA-mediated RNAi. To demonstrate RNAi of endogenous gene expression, we used heterozygous embryos of a knock-in mouse line expressing GFP from the Tis21 locus, a gene turned on in neuroepithelial cells that switch from proliferation to neurogenesis. GFP-directed esiRNA electroporated into neuroepithelial cells of such embryos blocked the GFP expression normally occurring on the onset of neurogenesis. Taken together, our data indicate that esiRNA delivered in a tissue-specific manner by topical injection followed by directed electroporation can efficiently silence endogenous gene expression in mammalian postimplantation embryos.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany.
authors.forename: Federico
authors.initials: F
authors.lastname: Calegari
authors.forename: Wulf
authors.initials: W
authors.lastname: Haubensak
authors.forename: Dun
authors.initials: D
authors.lastname: Yang
authors.forename: Wieland B
authors.initials: WB
authors.lastname: Huttner
authors.forename: Frank
authors.initials: F
authors.lastname: Buchholz
mesh_terms: D000818:Animals; D001921:Brain / Q000196:embryology* / Q000378:metabolism; D047108:Embryonic Development; D004722:Endoribonucleases; D005260:Female; D020868:Gene Silencing; D017930:Genes, Reporter; D016147:Genes, Tumor Suppressor; D049452:Green Fluorescent Proteins; D017874:Immediate-Early Proteins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D011247:Pregnancy; D034622:RNA Interference; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: C489435:Btg2 protein, mouse; D017874:Immediate-Early Proteins; D008164:Luminescent Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D049452:Green Fluorescent Proteins; D004722:Endoribonucleases; D001616:beta-Galactosidase