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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{alpha}. 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.





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