ASO-mediated RNA knockdown correlates with phenotype.

<p>A) The RNA level of the gene corresponding to each ASO was measured (compared to WT, normalized to <i>ef1a</i>) and correlated to the presence of a phenotype: green = reproduced published phenotype (in case of <i>MALAT1</i>: no mutant phenotype), orange = reproduced published phenotype in a smaller percentage of embryos, red = did not produce a phenotype. Injected amount of ASO per embryo: 50 pg <i>smad2</i> ASO, 50 pg <i>smad5</i> ASO, 50 pg <i>alk8</i> ASO, 50 pg <i>bmp7a</i> ASO, 150 pg <i>chordin</i> ASO, 100 pg <i>ntla</i> ASO, 50 pg <i>wnt11</i> ASO, 150 pg <i>tolloid</i> ASO, 100 pg <i>wnt5b</i> ASO, 100 pg <i>nacre</i> ASO, 150 pg <i>MALAT1</i> ASO. Error bars show standard deviation of the mean of 2 independent experiments (10 embryos each). B) ASO-generated phenotypes for <i>alk8</i>, <i>bmp7a</i>, <i>chordin</i>, <i>smad5</i> and <i>ctnnb2</i> (shown are 3 embryos representative of the different severities of <i>ctnnb2</i> ASO-induced phenotypes). C) ASOs only target the cognate RNA, and not unrelated RNAs. qPCR-based assessment of ASO specificity to their cognate target genes (10 embryos each). D) <i>dnd</i> ASOs block germ cell formation. Germ cells were labeled by injection of 80 pg <i>eGFP-nanos3’UTR</i> mRNA. Coinjection of 25 pg <i>dnd</i> ASO caused complete loss of green germ cells (white arrow).