Figure 3

Efficiency and specificity of miRNA knockdown using nanoparticle or liposome based antagomir transfection.

(a) Representative images of explanted E13.5 mandibles and SMGs (attached to tongue) in Trowell-type organ culture after 48 h of transfection of a fluorescently labeled antagomir (Antagomir-Cy3) using Liposome Forming Solution (LFS) or Nanoparticle Forming Solution (NFS) methods. Images correspond to 4–6 representative organ explants with experimental triplicates. Antagomir-Cy3 uptake was more efficient with NFS than LPS (for quantification of Antagomir-Cy3 uptake, see Supplementary Fig. 5). Scale bar: 100 μ. (b) Antagomirs targeting miR-590-5p and miR-200c-3p (Anta-590 and Anta-200c, respectively), were transfected into E13.5 mandible and SMGs (attached to tongue) in Trowell-type organ cultures. Molar and incisor tooth germs and SMGs were dissected after 24 h and miRNA expression assessed by qRT-PCR. Both NFS and LFS showed specific miRNA knockdown after transfection of the respective antagomir (middle and right graphs). Snord61 expression was used as an off-target control (left graph) and the qRT-PCR data were normalized to snRNA-U6 expression and plotted as percentage of expression compared to the control (represented here as 100% of expression, dotted red line). Expression differences in miRNAs after transfection of the corresponding antagomirs using LFS (pink squares) or NFS (blue triangles) are statistically significant (T-Student paired one tailed test), as follows: miR-200c-3p expression in molar tooth germs (p = 0.006), in incisors (p = 0.010) and SMGs (p = 0.011) using Anta-200c (graph in the middle); miR-590-5p expression in molar tooth germs (p = 0.010), in incisors (p = 0.006) and SMGs (p = 0.004), using Anta-590 (graph on the right). Snord61 expression did not change significantly in most treatments. For further data documenting the specifity of antagomir treatment, see Suppl. Figs 7 and 8.