Figure 3

Gene silencing by ex ovo RNAi is efficient. The efficiency of AX-1 downregulation was demonstrated in caudal sections taken from HH35 cerebella one day after injection and electroporation of dsRNA derived from AX-1 mixed with a plasmid encoding EGFP (50:1). (a) EGFP expression was used to identify the electroporated area of the cerebellum. (b,c) Expression of AX-1 was lost from many cells after ex ovo RNAi, resulting in a patchy appearance of the granule cell layer (b; c, open arrows). (d) In corresponding sections of a control embryo AX-1 staining appeared homogenous. (c) The number of EGFP-expressing cells (white arrows) was much smaller than the number of cells that failed to express AX-1 due to the molecular ratio of 50:1 for dsRNA and EGFP plasmid. Very few cells were yellow, indicating both EGFP and AX-1 expression (c, arrowhead). For quantitative analysis, 30-μm-thick sagittal sections were stained for AX-1. (e) EGFP expression was used to identify the electroporated area of the cerebellum. Staining intensities were compared between experimental (e,f) and control (EGFP) embryos (h) at HH35, one day after electroporation. (f,i) On average, the injection and electroporation of AX-1 dsRNA reduced the AX-1 protein level in the transfected area by 61.3 ± 9.4% (n = 4 embryos; ***P < 0.0001 for comparison with both control groups). (g) As expected, there was no change in AX-1 protein levels in the untransfected part of the cerebellum of embryos treated with dsRNA. (h,i) AX-1 expression in transfected areas of embryos treated with the EGFP plasmid alone did not differ from untreated control embryos (not shown; i). The ratio of AX-1 staining was 3.4 ± 2.2% (n = 5) for EGFP-control embryos and 2.3 ± 0.9% (n = 4) in untreated control embryos. The insert in (e) shows a low magnification image of the section. The box corresponds to the high magnification image shown in (e). PS, pial surface. Bar: 50 μm in (a-d), 200 μm in (e-h).