Fig. 3. Depletion of XPACE4 using an antisense oligo approach. (A) Real-time RT-PCR analysis of oocytes (oo) injected with 5-10 ng unmodified antisense oligos shows depletion of maternal XPACE4 mRNA to variable degrees. (B) Phosphorothioate modified AS-5 oligo (AS-5MP) depletes XPACE4 mRNA without affecting the mRNA levels of a closely related maternal convertase Furin (XFurA) as analyzed by real-time RT-PCR. (C) XPACE4 mRNA levels in control and XPACE4-depleted embryos: XPACE4-depleted embryos (AS-5MP+) are generated by the host transfer technique and are compared with sibling uninjected control embryos at blastula (7 and 8) and gastrula (10, 11, 12) stages. Real-time RT-PCR shows that XPACE4 is depleted down to 5% and it does not reach the control levels (AS-5MP-). (D) Antisense oligo depletion of XPACE4 reduces signaling activity of Xnr1 in oocytes. A schematic presentation of the paracrine assay is shown at the top. Animal caps co-cultured with uninjected control oocytes have very low or no detectable levels of organizer genes chordin (Chd), goosecoid (Gsc), mesodermal gene Xbra or endodermal gene XSox17. All of these genes are induced when caps are co-cultured with control oocytes injected with Xnr1 mRNA. Animal caps co-cultured with XPACE4-depleted oocytes injected with Xnr1 mRNA show a reduction in the expression levels of these mRNAs. Sibling whole embryo (WE) is used for the dilution series and the quantification in real-time RT-PCR.