Effective dsRNA delivery and EGFP knock-down by electroporation. (A) Representative dark-field projections of the rendered z-stack of x-y confocal sections of blastocysts electroporated with AF594-labelled dsRNA (540 bp) after 24 h of culture. One hundred percent of the blastocysts incorporate the dsRNA and an average of 90% of the blastomeres in each embryo (minimum 70%) are targeted. Left, the ICM is viewed from the top; right, the ICM is viewed laterally. (B) Representative confocal bright-field images (top) and dark-field projections of the rendered z-stack of x-y sections (bottom) of HB2-EGFP embryos electroporated with GFP dsRNA and LacZ dsRNA (control) at the 8-cell and blastocyst stages, followed by 24 hours in culture (24 AE) and in utero development until the indicated stages (E4.3 and E5.5). The reduction of GFP fluorescence intensity following RNAi was similar among both the ICM cells (white arrowheads) and trophectoderm cells (red arrowhead). Likewise, despite the difference in fluorescence intensity between the epiblast cells and the surrounding extraembryonic tissues, GFP fluorescence was reduced to a similar degree in both tissues (white arrow and red arrow, respectively). (C) Average levels of fluorescence intensity of the main cell lineages of GFP dsRNA-electroporated blastocysts (24 h AE), and post-implantation embryos at the indicated stages, shown as a percentage of the intensity levels of control (LacZ dsRNA-treated) embryos. Bar colors as follows: cultured blastocysts (n = 9): brown, ICM; dark red, trophectoderm; E4.3 and E5.5 embryos (n = 8 and n = 7, respectively): blue, epiblast; light blue, visceral endoderm and extraembryonic ectoderm. Note that the reduction of fluorescence intensity among the different lineages at any given stage of development is very similar. Standard deviation bars are indicated. (D) Average GFP mRNA levels (GFP/GAP3DH ratio) of embryos electroporated with GFP dsRNA shown as a percentage of those of LacZ dsRNA-treated controls. Pale blue, 15 dsGFP-electroporated embryos cultured in vitro for 24 h (control embryos: 15, dark blue). Light blue, 4 and 3 dsGFP-electroporated embryos recovered at E4.3 and E5.5, respectively, following in utero development (control embryos: 8, dark blue). Standard deviation bars are indicated.
Soares et al. BMC Developmental Biology 2005 5:28 doi:10.1186/1471-213X-5-28